Cytek® Aurora User’s Guide

Copyrights
© 2021, Cytek Biosciences, Inc. All rights reserved. No part of this publication may be reproduced, transmitted,
transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any
means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from
Cytek Biosciences.

The information in this guide is subject to change without notice. Cytek Biosciences reserves the right to change its
products and services at any time to incorporate the latest technological developments. Although this guide has been
prepared with every precaution to ensure accuracy, Cytek Biosciences assumes no liability for any errors or omissions,
nor for any damages resulting from the application or use of this information. Cytek Biosciences welcomes customer
input on corrections and suggestions for improvement.

Trademarks
© 2021 Cytek, the Cytek logo, SpectroFlo, Similarity and Complexity are trademarks of Cytek Biosciences, Inc. All other
service marks, trademarks and tradenames appearing in this guide are the property of their respective owners.

FCC Information
WARNING: Changes or modifications to this unit not expressly approved by the party responsible for compliance could
void the user's authority to operate the equipment.

NOTICE: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to
Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when
the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio
frequency energy and, if not installed and used in accordance with the instruction manual, can cause harmful
interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful
interference in which case the user will be required to correct the interference at his or her own expense.

Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits.

This Class A digital apparatus meets all requirements of the Canadian Interference-Causing Equipment Regulations.

Cet appareil numérique de la classe A respecte toutes les exigences du Réglement sur le matériel brouilleur du Canada.

CDRH Information
Class I laser product.

Regulatory Information
For Research Use Only. Not for use in diagnostic or therapeutic procedures.

History

Revision Date Change

52-70001-0A 10/2017 Initial release

52-70001-0B 1/2018 Added details to safety, acquisition, and unmixing

52-70001-0C 1/2021 Added Loader, yellow-green laser, keywords, SSC off blue laser, label
and lot-specific unmixing, and group-specific unstained controls

52-70001-0D 3/2019 Added ultraviolet laser

N9-20006 Rev. A 6/2021 Updated SpectroFlo QC beads lot number, corrected laser
bandwidth table, changed user’s guide part number

N9-20006 Rev. B 10/2019 Updated to SpectroFlo v2.2 to include reference control QC

(benchmarks), clog and bubble detection

N9-20006 Rev. C 6/2021 Updated to SpectroFlo v3.0, added information on new loader

N9-20006 Rev. D 7/2021 Updated steps of some workflows, updated screenshots, and
corrected typos.
 Contents

Chapter 1: Introduction 7
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
General Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Biological Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Chapter 2: Overview 11
Cytometer Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Fluidics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Software Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Get Started Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
About Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
About Worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Chapter 3: Startup & Shutdown 25

Filling the Sheath and Emptying the Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Filling the Sheath . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Emptying the Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Starting Up the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Shutting Down the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Chapter 4: QC & Setup 29

Daily QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Performing Daily QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
QC Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Reference Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Running Reference Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Updating Reference Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Levey-Jennings Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Gain Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Alarm Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

iii
Chapter 5: Acquisition 43
Raw vs Unmixed Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Unmixing and Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Setting Up an Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Acquisition Experiment Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Experiment Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Creating a Default Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Creating a New Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Chapter 6: Unmixing and Compensation 61

Spectral Unmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Understanding Full Spectrum Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Unmixing Workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Unmixing Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Live Unmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Post-Acquisition Unmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Virtual Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Chapter 7: Library, Preferences, and Users 79

Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
QC Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Fluorescent Tags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Keywords . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Spillovers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
User Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Loader Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Worksheet Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Experiment Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Backup & Restore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Acquisition Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Worksheet Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Plot Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Gates Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Statistics Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Fonts Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Annotation Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Notifications Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Storage Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
QC Setup Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Cytometer Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Users Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

iv Aurora User’s Guide

 Global Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Managing Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Use Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
User Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
User Policy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Chapter 8: Loader 109

Loader Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Using the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Enabling the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Loading a High-Throughput Sample Carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Loader Settings in the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Group Hierarchy/Plate Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Loader Acquisition Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Loader Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Experiments in Plate Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Creating Groups When Using the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Defining Loader Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Calibrating a Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Calibrating the SIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Chapter 9: Maintenance 121

Maintenance Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Scheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Unscheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
SIT Flush . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Purge Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Clean Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Long Clean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Fluidics Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Fluidics Shutdown Using Individual Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Fluidics Shutdown Using a Tube Rack (ASL only) . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Cleaning the External Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Inspecting the Fluidics Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Replacing the Sheath Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Replacing the SIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Chapter 10: Troubleshooting 131

Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Chapter 11: Glossary 135

Chapter 12: Specifications 139

Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

v
 Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Fluorescence and Scatter Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Installation Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Supported Carrier Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Chapter 13: Supplies and Replacement Parts 143

Index 145

vi Aurora User’s Guide

 1
Introduction

About This Guide

This guide provides information on the Aurora flow cytometer, daily workflow, SpectroFlo®
software features, instrument maintenance, and cytometer specifications. It also includes
troubleshooting tips and service information.

Safety

Safety Symbols
The Aurora is intended for research use only; not for diagnostic or therapeutic procedures.
The following table lists symbols used throughout this guide.

Symbol Meaning

Caution: hazard or unsafe practice that could result in material damage,

data loss, minor or severe injury, or death

Risk of electric shock

Biological risk

Laser radiation

General Safety
• Read all safety instructions completely before using the equipment. Keep the instructions in a
safe place.
• Follow all instructions when operating the instrument.
• If the equipment is used in a manner not specified by the manufacturer, the protection
provided by the equipment may be impaired.
• Do not place any object on top of the instrument.

Chapter 1: Introduction 7
• Do not block any ventilation openings.
• Do not place the unit near any heat sources such as radiators, heat registers, stoves, or other
devices (including amplifiers) that produce heat.
• Use only attachments/accessories specified by the manufacturer.
• Use only with the cart, stand, tripod, bracket, or table specified by the manufacturer or sold with
the equipment. When a cart is used, use caution when moving the cart/equipment combination
to avoid injury from tipping over.
• Unplug the instrument when it will not be used for long periods of time.
• Refer all servicing to qualified service personnel. Service is required when the unit has been
damaged in any way, such as: if the power-supply cord or plug is damaged, if the unit is
dropped, if liquid is spilled onto the unit or objects fall into the unit, if the unit is exposed to rain
or moisture, or if the unit does not operate properly.
• Do not expose the instrument to temperatures outside the range of 15°C to 28°C.
• Concentrations of sodium hypochlorite (bleach) higher than 10%, as well as other cleaning
agents, can damage the instrument. Use a 10% solution of household bleach to clean, where
indicated. A 10% bleach solution is prepared by adding 1 part household bleach to 9 parts
deionized water.
 NOTE: Household bleach contains 5–7% sodium hypochlorite.
• Before turning on the cytometer, visually inspect all containers. Wear appropriate personal
protective equipment (PPE), including, but not limited to, gloves, eyewear, and lab coat.
• Purge the sheath filter if air bubbles are visible in the sheath filter, or if the plenum or sheath
container have run dry.
• Fill the sheath container as needed. Never use tap water as sheath solution. Never use
surfactant-based sheath solutions.
• Do not run bleach or detergent through the sheath filter. It is difficult to remove cleaning
solutions from the sheath filter.
• Check the cytometer periodically for fluid leaks or crimped lines. If evidence of a leak is
detected, contact Cytek Technical Support immediately. Do not attempt to repair the
instrument.
• When performing Daily QC, always select the current bead lot number.

Electrical Safety
• Do not place liquids on top of the instrument. Any spill into the ventilation openings could
cause electrical shock or damage to the instrument.
• Do not use this equipment near water.

WARNING: To reduce the risk of fire or electric shock, do not expose this apparatus to
rain or moisture.

• Use only the power supply cord specified by the manufacturer. The power cord of the unit is
equipped with a 10A three-prong power plug. Do not remove the ground pin of the power plug
under any circumstances. Make sure the plug is securely plugged into the power outlet to
prevent fire. If the power supply cord needs to be replaced, the cross section area of the
conductor should be at least 16 AWG. This is to prevent electric fire or shock.
• Use only the fuse specified by the manufacturer. The fuse is 250 VAC, 5 A, size 5 x 20 mm.

8 Aurora User’s Guide

• Protect the power cord from being tread upon or pinched, particularly at the plug and the point
where it emerges from the equipment. Be sure that the power outlet is located near the
equipment so that it is easily accessible.

Biological Safety
• Empty the waste container when filling the sheath container or as needed to prevent leakage or
system damage. Take care to avoid damaging the fluid level sensor in the waste tank.
• Follow local, state, and national regulations when disposing of waste.
• Biological samples are potentially dangerous and/or life threatening. Adhere to proper
handling procedures for samples and reagents. Wear appropriate personal protective
equipment (PPE), including gloves, eyewear, and lab coat.
• Any instrument surface in contact with biological specimens can transmit potentially fatal
disease. Use universal precautions when cleaning the instrument or replacing parts.

Laser Safety
The Aurora is a Class 1 Laser Product and complies with the US FDA Center for Devices and
Radiological Health 21 CFR 1040.10 and 1040.11 except for deviations pursuant to Laser Notice No.
50, dated June 24, 2007. No laser radiation is accessible to the user during normal instrument
operation.
The lasers are fully contained within the instrument and do not require any special work area
safety except when service procedures are being performed. These procedures are performed only
by Cytek service personnel.

CAUTION: Modification or removal of the optics covers or laser shielding could result in
exposure to hazardous laser radiation. To prevent damage to skin and eyes, do not
remove the optics covers or laser shielding, or attempt to service the instrument where
laser warning labels are attached.

Use of controls or adjustments or performance of procedures other than those specified in this
user’s guide may result in hazardous radiation exposure.
To prevent exposure to laser radiation:
• Do not defeat any safety interlocks on the instrument.
• Do not use controls, make adjustments, or perform procedures other than those specified in
this user’s guide.
• Do not attempt to perform service procedures on the lasers.

Chapter 1: Introduction 9
Technical Support
For instrument support within the US, call 1-877-92-CYTEK (1-877-922-9835).
Outside the US, call 1-510-657-0102. In Europe, call +31207653440.
Email Cytek at [email protected].
Visit our website, www.cytekbio.com, for up-to-date contact information.
When contacting Cytek, have the following information available:
• Serial number
• Any error messages
• Details or screen shots of recent system performance
• SpectroFlo software version and system firmware version, located in the Help module

10 Aurora User’s Guide

 2
Overview

Aurora System
The Aurora system consists of the Aurora flow cytometer and a computer workstation running
SpectroFlo software for sample acquisition and data analysis. The system is intended to be used for
analyzing cells in the fields of immunology, biochemistry, biology, oncology, hematology, virology,
and pathology research.
The cytometer is an air-cooled, compact benchtop instrument. It is equipped with up to five lasers,
64 detection channels for fluorescence, and three channels for scatter (blue laser FSC, blue laser
SSC, and violet laser SSC). Sheath and waste fluids are contained in either 4-L tanks, included with
the system, or 20-L cubitainers. Software indicators notify you when the sheath is getting low or
the waste is getting full. The fluidics system includes a plenum for storing sheath, allowing you to
fill the sheath tank during operation.
Optional high-throughput sample Loaders are available to automate sample delivery and
acquisition. The loaders are compatible with 96-well plates. The new Automated Sample Loader
(ASL) offers added compatibility for 96-well deep-well plates and 40-tube racks. See the “Loader”
chapter for more information.
The workstation is a dedicated USB-compatible PC with monitor, keyboard, and mouse. It runs
Microsoft® Windows® 10 Pro with a 64-bit operating system, which is required for SpectroFlo
software.

Fluidics tanks Aurora instrument Workstation

Chapter 2: Overview 11
Cytometer Overview
Solid-state lasers transmit light through a flow cell where particles in suspension are focused single
file for interrogation by the laser. Proprietary, high-sensitivity, semiconductor detector APD arrays
are equipped with up to 16 channels per laser to capture the emission spectra of dyes that emit in
the 365 to 829 nm wavelength range. The resulting fluorescence and scatter signals are then
collected and converted into electronic signals. On-board electronics convert these signals into
digital data that can be acquired and recorded on the workstation.
The cytometer power button is located on the left side panel in the upper right area. When the
cytometer is powered on, the power button is illuminated.
The front panel opens on hinges to the left to reveal the fluidics system. The SIT door to the right of
the front panel opens to reveal the sample injection tube (SIT) assembly. The top cover opens to
reveal the optics plate.

Front of Cytometer

SIT door

Do not place any object on top of the instrument.

Do not place liquids on top of the instrument. Fluid leaking into the cytometer could
cause electrical shock or damage to the instrument.

12 Aurora User’s Guide

Back of Cytometer
Allow 20.0 cm (8 in) between the back of the cytometer and the wall for proper ventilation without
an air duct. Allow 10.0 cm (4 in) with an air duct.

USB connection to workstation

main power switch

power cable

Fluidics System

Sample Injection Port and Sample Injection Tube

Sample, contained in a standard 12 x 75-mm tube, enters the cytometer through the sample
injection tube (SIT) that is contained within the sample injection port (SIP). The 12 x 75-mm sample
tube snaps into place under the SIP requiring no additional tube retention support. The SIT extends
from the SIP during acquisition and retracts when the cytometer is not acquiring.
When using the optional Loader, a 96-well plate, 96-well deep-well plate, or 40-tube rack can be
used for sample delivery in place of individual 12 x 75-mm sample tubes. See the Loader chapter
starting on page 109 for more information.

SIP

SIT

Chapter 2: Overview 13
Fluid Containers
The Aurora draws sheath solution directly from a 20-L sheath cubitainer or the 4-L sheath tank
provided by Cytek. It expels waste into an empty 20-L cubitainer or the 4-L waste tank provided by
Cytek.
The fluidics tanks are contained in a holding reservoir located on the left side of the cytometer. The
4-L tank with the transparent fluidic line is for sheath solution. The 4-L tank with the orange fluidic
line is for waste.

waste

sheath

Fluid Flow
The Aurora fluidics are driven by vacuum. An accumulator vessel is the source of vacuum for the
system. Sheath solution is drawn into and stored in the sheath plenum before passing through a
sheath filter, where debris and contaminants are removed. Before reaching the flow cell, the
sheath stream passes through a degasser, which removes air bubbles. After passing the laser
interrogation point, the combination of sheath solution and sample travels to the waste container.
Sheath and waste fluid levels are monitored by sensors. The waste level sensor is located
underneath the waste tank cap. The sheath level sensor is located underneath the sheath plenum
cap. Both sensors are monitored by the software.

14 Aurora User’s Guide

Fluidics Components
The following figure shows the fluidics components.

1
2

3
4

6
7

The following table describes the fluidics components.

No. Component Description

1 Plenum pump Pulls sheath from the sheath tank to fill the plenum
2 Vacuum pump Maintains the vacuum in the accumulator
3 Plenum Storage vessel for sheath fluid before it flows to the sheath filter
4 Degasser Removes air bubbles from the sheath fluid
5 Sheath filter quick Sheath filter fluid input, fluid output, and vent line quick-connects
connects (x3)
6 Sheath filter Filters debris and particles from the sheath fluid
7 Accumulator Vacuum source for the fluidics system

Optics
Unlike conventional flow cytometers that direct specific bandwidths of fluorescence light into
discrete detectors or photomultiplier tubes (PMTs), the Aurora uses a solid-state, multi-channel,
narrow-beam detector array for each laser. Each array can be configured with up to 16 detectors
that are used to capture a part of the emission spectrum from each particle passing through the
laser beam. The detector channels from up to five lasers are used to capture the entire emission
spectra from each fluorescent-labeled particle. Spectral deconvolution (unmixing) algorithms
calculate the contribution of the known individual fluorophore’s spectra to the total collected signal.

Chapter 2: Overview 15
For excitation, a proprietary flat-top laser design enables a constant power distribution across the
width of the sample core stream.

Optical configurations are as follows:

Laser Excitation Channels for detection Detector names

Ultraviolet 355 nm 16 UV1–UV16

Violet 405 nm 16 V1–V16
Blue 488 nm 14 B1–B14
Yellow-Green 561 nm 10 YG1–YG10
Red 640 nm 8 R1–R8

 NOTE: Different laser configurations are available. Yellow-Green and Ultraviolet lasers are not
available on the Northern Lights cytometers.
The wavelengths detected by each detector (channel) increase across the array. See the table on
page 75 for details.

16 Aurora User’s Guide

Software Overview
SpectroFlo software allows you to acquire and analyze samples and adjust instrument settings.
Once you log into the software, a Get started menu appears.

Get Started Menu

The Get started menu provides six modules that allow you to perform various functions. These
same six modules are also accessible across the upper-right corner of each module screen.

The following table describes options in the Get started menu.

Module Description

QC & Setup Daily QC ensures that the instrument is in optimal condition for use. Run
SpectroFlo QC Beads daily to assess system performance and to adjust settings
to account for day-to-day variation. Levey-Jennings reports keep track of trends
in system performance. Setup allows you to create reference controls. See “QC &
Setup” on page 29 for information.
Acquisition The Acquisition module allows you to create experiments to acquire and analyze
data. Experiments can be created through a guided wizard or created from
previously saved templates. See “Acquisition” on page 43 for information.
Extra Tools Here FCS files can either be unmixed or compensated using virtual filters. This is
where you can Remove FCS parameters such as Area, Height, and Width as well.
See “Unmixing and Compensation” on page 61 for information.
Library The library allows you to store experiment templates, worksheet templates, user
settings, fluorescent tags, SpectroFlo QC bead information, label information,
keywords, and Loader settings. See “Library” on page 79 for information.
Preferences Software preferences can be changed to customize the software. Default plot
sizes, fonts, gate colors, print layouts, statistics box table option, and more can
all be changed in the Preferences. See “Preferences” on page 87 for information.

Chapter 2: Overview 17
 Module Description

Users The Users module contains user management options and administrative
controls. See “Users” on page 101 for information.

About Experiments
The Acquisition module provides the tools necessary to acquire data, such as the acquisition
controls used to start, stop, and record data, and the instrument controls used to set the threshold
and adjust the detector gains. See “Experiment Display” on page 44 for more information on these
controls. Experiments contain the fluorescent tags and labels used in the experiment, the stopping
criteria, and the groups of tubes/wells run, which can include the reference control group. You can
create groups for your samples, if you wish, to conveniently organize samples by type or staining
panels, for example.

Opening an Experiment
When you click Acquisition from the Get started menu, the Acquisition Experiment menu (below) is
displayed, allowing you to open a default or template experiment, create a new experiment, or
import an experiment. A wizard walks you through the steps to create a new experiment.
 NOTE: By default the Acquisition menu in the left pane is collapsed, showing only the icons for
Experiment, Worksheet, Cytometer, and Plate Calibration. To expand the menu to show the labels,
click the arrows (>>) at the bottom of the pane. The red boxes in the figure illustrate the expanded
view and what you see if you are a User or an Administrator.
User Administrator

Experiments can be created using several different methods. The following table describes the
options in the Acquisition Experiment menu:

Method Description

Default Opens a new experiment with one group containing one tube and a set of labels
and fluorescent tags in a default experiment worksheet template. The default
experiment is user configurable. It is the quickest way to begin sample
acquisition.
New Opens the New Experiment Wizard to guide you through creating an
experiment.
Template Allows you to select from a list of saved experiment templates (see page 19).

18 Aurora User’s Guide

 Method Description

Import Imports an experiment ZIP file that was exported.

My Allows you to select from a list of saved experiments.
Experiments NOTE: Original experiments can be duplicated without data, which is equivalent
to opening an experiment template. Right-click an experiment in My
Experiments and select Duplicate (with or without data). You can also choose
Duplicate with Reference group data.

Completed experiments can be accessed through the My Experiments option in the Acquisition
Experiment menu. Use the column headers to sort the list of experiments. For every tube recorded,
two FCS files are saved, one raw and one unmixed. Use My Experiments to open experiments you
already ran, as you may want to review the data or acquire more samples. You can also export
experiments from My Experiments (below). A ZIP file is exported, containing all the raw data files, if
applicable (and unmixed files for unmixed experiments), as well as the worksheet templates and
experiment template.

FCS Files
FCS files generated from an experiment are stored in the Export folder by default, or the folder you
set as the default. See “Storage Preferences” on page 97 for information. Experiments can contain
the following types of FCS data files for each tube run:
• raw data files only (for samples that were acquired in an experiment)
• raw data files + unmixed data files (for samples that were acquired and unmixed live during
acquisition)
• unmixed data files only (for samples that were unmixed post acquisition)

Experiment Templates
Use the Save As option above the experiment’s tube/group (hierarchy) list to save the current
experiment as a template, which can then be used for running similar experiments. Experiment
templates include fluorescent tags used in the experiment, reference controls, groups/tubes,
labels, worksheets, and stopping criteria. Templates are saved in the library. To open and use a

Chapter 2: Overview 19
template, select Template from the Acquisition Experiment menu. See “Experiment Templates” on
page 85 for more information on experiment templates.

20 Aurora User’s Guide

About Worksheets
Worksheets are used to visualize the data in the experiment. Each experiment requires at least one
worksheet. A worksheet allows you to view the data in plots during acquisition, as well as perform
analysis functions. Worksheets contain the tools necessary to create plots, gates, annotations,
statistics, and the population hierarchy. Worksheets are saved with the experiment and can be
saved separately and reused across experiments.
Two types of worksheets are available—worksheets for raw data and worksheets for unmixed data.
You must select the appropriate worksheet to view the corresponding type of data. When viewing
raw data, the parameters on the plots in a raw worksheet reflect the channel names, for example,
B1-A, R1-A, V1-A. When viewing unmixed data, the parameters on the plots in an unmixed
worksheet reflect the fluorescent tags, for example, PerCP-A.

Opening a Worksheet
To select a worksheet, click Worksheet in the Acquisition menu. The Select a worksheet menu
appears (below). You can open a new raw or new unmixed worksheet. These worksheets open with
a single FSC vs SSC plot. Use the worksheet tool bar to add plots and other elements. All
worksheets are saved as template files (WTML) and can be opened using the Open Worksheet
Template option. You can also import worksheets that were exported, as well as save, print, and
save a PDF.

The following table describes the options in the Acquisition Worksheet menu:

Method Description

Default Raw Opens a default raw worksheet that can be used for experiments where
reference controls will be acquired. Do not overwrite this worksheet. Always
use Save As to save this worksheet with a new name.
New Raw Opens a new raw worksheet.
New Unmixed Opens a new unmixed worksheet.

Chapter 2: Overview 21
 Method Description

NxN Plots Uses the fluorescent tags selected when setting up the experiment to create a
Unmixed worksheet with multiple plots, displaying each fluorescent parameter against
each other. This worksheet allows you to check for potential unmixing errors
and correct as needed.
Open Allows you to select from a list of saved worksheet templates. A default raw
Worksheet and default unmixed worksheet are provided.
Template
Import Imports a worksheet template that was exported.
Save, Save As, Saves the worksheet, saves the worksheet with a new name, prints the
Print, Save PDF worksheet, saves a PDF of the worksheet.

You can have multiple worksheets open at a time. The currently displayed worksheet appears with
a blue line under the worksheet name. Because you can select different worksheets for different
groups or tubes in an experiment, each tube will have a worksheet associated with it.

22 Aurora User’s Guide

Each user can define a default raw worksheet and a default unmixed worksheet. Open the default
worksheet and set it up for your experiment, then select Save As to save this worksheet with a new
name. The worksheet will be available to select when you create an experiment. You can use this
worksheet, open a template worksheet, or create a new worksheet.
All worksheets are saved in the library. See “Worksheet Templates” on page 84 for more
information on worksheet templates.

Chapter 2: Overview 23
24 Aurora User’s Guide
 3
Startup & Shutdown

Filling the Sheath and Emptying the Waste

The color-coded sheath and waste quick-connects and the waste level sensor connector are
located at the lower-left corner of the front panel.

sheath line quick-connect

waste level sensor

waste line quick-connect

 NOTE: We recommend using the 20-L cubitainers instead of the 4-L tanks for systems with a
Loader. If using the original AMS, place fluid tanks on the floor beneath the instrument, otherwise
fluid can backflush into the wash well. If your AMS has been upgraded to version 1.5, or you are
using the ASL, this precaution is no longer necessary.

Filling the Sheath

Fill the sheath container with manufacturer-provided sheath solution, MilliQ™ water, phosphate-
buffered saline (PBS), or deionized (DI) water.
Sheath can be drawn from either the supplied 4-L sheath tank or directly from a 20-L cubitainer.
Sheath solution can be added to the sheath container while the instrument is running.

Before turning on the cytometer, visually inspect all containers for leaks or cracks. Wear
the recommended PPE such as protective gloves, eyewear, and lab coat.

Fill the sheath container as needed. Use only the appropriate sheath solution. Never
use tap water or surfactant-based sheath solution.

Chapter 3: Startup & Shutdown 25

Filling Sheath into a Cytek 4-L Sheath Tank or a 20-L Cubitainer
1 Remove the sheath fluidics line cap from the cubitainer or sheath tank lid from the sheath tank.

2 Add the appropriate sheath solution.

3 Replace the fluidics line cap or sheath tank lid. Do not over-tighten.

4 If the cytometer is powered on and the software is connected, verify that the software sheath
indicator is green.

Emptying the Waste

Waste can be collected into either the supplied 4-L waste tank or directly into an empty 20-L
cubitainer.

Empty the waste container when filling the sheath container, or as needed to prevent
leakage. The software indicator for waste will be yellow or red when the container needs
to be emptied. Take care to avoid damaging the fluid level sensor in the waste tank.

Biological samples are potentially dangerous and/or life threatening. Adhere to proper
handling procedures for samples and reagents. Wear PPE such as protective gloves,
eyewear, and lab coat during this procedure.

Always treat the contents of the waste container with household bleach (10% of the
total volume). Contents of the waste container may contain biohazardous material.

Removing Waste from a Cytek 4-L Waste Tank or a 20-L Cubitainer

1 Disconnect the waste line orange quick-connect from the cubitainer cap or 4-L waste tank.
Disconnect the waste level sensor.
The waste level sensor connector for the cubitainer is on the cubitainer cap. The waste level
sensor connector for the 4-L tank is on the front of the cytometer.

2 Remove the waste cap from the cubitainer or the lid from the 4-L waste tank, taking care not to
damage the liquid level sensor.

3 Dispose of the waste according to local, state, and national regulations.

4 Add 2 L of undiluted bleach to the waste cubitainer, or 400 mL of bleach to the waste tank.

5 Replace the waste cap/lid to the container. Hand-tighten the cap/lid until it is fully closed.

6 Reattach the waste line and level sensor line to the cap/lid and front of the cytometer.

7 If the cytometer is powered on and the software is connected, verify that the software waste
indicator is green.

26 Aurora CS User’s Guide

Starting Up the System
1 Turn on the workstation, then turn on the cytometer.
 NOTE: For manual tube configuration, ensure that a tube containing 1 mL of deionized (DI)
water is loaded on the SIP before launching SpectroFlo software. The tube is required for the
SIT depth calibration and to flush liquid through the flow cell to remove bubbles that may have
formed. For the ASL, the software will prompt you to place a tube in the loader with DI water in
the rack.

2 Launch SpectroFlo software and log in by entering your user name and password and clicking
SIGN IN.

You can start typing your user name to display a list of names beginning with the letter(s) you
type.
The cytometer initialization procedure begins. Sheath fluid is flushed through the fluidics lines
to prevent any saline buildup, and the system calibrates the SIT depth.

3 Select Acquisition from the Get started menu to launch the startup wizard.

4 Check the status indicators in the lower-right corner of the screen.

• Ensure the indicator for Connected is a green checkmark. It may take a few minutes for the
indicators to update.

If the indicator shows the instrument is not connected, check to ensure that the USB
connection between the cytometer and workstation is plugged into the appropriate ports.
See “Back of Cytometer” on page 13.

Chapter 3: Startup & Shutdown 27

 • Ensure the status indicators for sheath and waste are green before proceeding.

Fluid Indicator Meaning What to do

Yellow sheath Sheath tank is low. Acquisition will Fill the sheath tank (see “Filling the
continue for 5 minutes before the Sheath” on page 25).
sheath is empty.
Red sheath Sheath tank is empty.
Yellow waste Waste tank is nearing capacity. Empty the waste tank (see
“Emptying the Waste” on page 26).
Red waste Waste tank is full.

5 Wait 30 minutes before running samples. We recommend running a tube of DI water during the
warm-up period. You cannot record a file until the 30 minute warm-up period is complete.

 NOTE: Administrators can select Enable data collection during system warm up under Preferences
> Cytometer. This bypasses restrictions imposed by default during instrument warm-up, such as
data acquisition and Daily QC.

Shutting Down the System

Run the shutdown procedure at the end of each day that you use the instrument.
The shutdown procedure flushes the flow cell and sample lines with 10% bleach solution, DI water,
30% Contrad 70, and DI water. The procedure takes approximately 5 minutes.
To shut down the system:
1 Follow the Fluidics Shutdown procedure. See “Fluidics Shutdown” on page 124.

2 When the shutdown procedure is complete, exit SpectroFlo software by clicking the X in the
upper-right corner of the application window.

3 Turn off the cytometer and workstation.

28 Aurora CS User’s Guide

 4
QC & Setup

Daily QC
Run Daily QC using SpectroFlo QC beads prior to acquiring samples to ensure that the cytometer is
performing optimally. Daily QC assesses the instrument’s optical alignment and the system
performance drift by measuring rCVs and gains needed to place the beads at the target locations
for each detector. During QC, laser delays and area scaling factors are optimized and gain settings
are adjusted to account for day-to-day instrument variability. Upon completion of Daily QC, a QC
report is generated. QC reports can be reviewed under the Reports tab.
Performance can be tracked and charted over time in the Levey-Jennings tab. The software can be
configured to display a warning if the QC result on the QC report exceeds user-defined criteria. See
“Alarm Ranges” on page 41.

Performing Daily QC
1 Allow 30 minutes to pass after turning on the system to ensure the optics compartment is
warmed up. The system has grayed out the run button in QC & Setup until the warm-up time is
complete.
The warm-up time is displayed in the status indicators in the lower-right corner of the software.

2 Prepare SpectroFlo QC beads (1 drop of beads in 0.3 mL of sheath solution) in a tube or plate
well.

 NOTE: Always prepare beads in the same solution used for the sheath solution on the
instrument. The bead diluent and instrument sheath solution must match. If you use DI water
to prepare the beads, the beads will begin to degrade within a few hours. Do not reuse the
beads prepared in DI water.
The SpectroFlo QC beads are 3-µm hard-dyed, polystyrene beads that have a single
fluorescence intensity. They can be excited by each laser and emit fluorescence in all detector
channels.

Chapter 4: QC & Setup 29

3 Select QC & Setup from the Get started menu.

4 Select the current bead lot from the Bead Lot menu.

Each time you open a new lot number of SpectroFlo QC beads you must import the bead lot ID
into the library so it is accessible when you run QC. Bead lot files can be downloaded from the
Resources section at http://www.cytekbio.com.

Different bead lots have different fluorescence intensities. Always select the correct
bead lot when performing Daily QC.

5 Ensure the correct Carrier Type is selected.

6 Start acquisition:

• Tube: Load a 12 x 75-mm tube of the beads onto the SIP. Select Start to begin acquisition.

30 Aurora User’s Guide

• Loader: Ensure the correct plate type is selected (96 U-, V-, Flat-Bottom, deep well, or 40-tube
rack). Click in the plate to select the well where the QC beads are located. Well A1 is selected
by default, but you can choose any well. Select Eject, load the plate onto the plate stage, then
click Load followed by Start to begin acquisition.

As the instrument begins acquiring the QC beads, they appear in the scatter plot. The laser
delays are initially set to 0, then optimized thereafter. The performance measurements are
established and compared to the pass/fail criteria (see “Pass/Fail Criteria” on page 32).
The procedure takes approximately 3 to 5 minutes to complete. Once acquisition is complete,
two SIT Flushes are automatically performed to clear the beads from the sample line.
The following message is displayed when Daily QC passes. To view the QC report, click View
Report (see “QC Report” on page 32).

Chapter 4: QC & Setup 31

 If QC fails, remove the tube or plate and follow the guidelines that appear. The recommended
solution will vary depending on the reason for the failed test.

You are now ready to run reference controls, if applicable, or acquire samples.

QC Report
At the completion of Daily QC, a QC report is generated. The report includes the following sections:
• The header section contains the Pass/Fail status of the run, name of the instrument, instrument
configuration, date the Daily QC was run, user who ran the Daily QC, instrument serial number,
and SpectroFlo QC bead lot and expiration date.
• The results section contains the gain, gain change, median fluorescent intensity of the QC bead,
%rCV, and a pass/fail indicator for each detector channel. The center wavelength of the detector
is shown in parentheses next to the detector name.
• The Laser Settings section contains the laser delays for all non-primary lasers, and area scaling
factors for all lasers and the FSC detector.

Pass/Fail Criteria
The pass/fail criteria are the following:
• %rCV must not exceed 6% for the FSC channel
• %rCV must not exceed 8% for the SSC and SSC-B channels
• %rCV must not exceed 6% for the third channel of each laser (V3, B3, R3, YG3, and UV3)
• % delta gain change for all channels must not exceed 100% from the last Daily QC run
performed by Cytek Service personnel.
QC reports are automatically exported as CSV files to the Setup folder (C:\CytekbioExport30\Setup).
The number of reports listed in the Reports screen can be set in the Preferences. See “QC Setup
Preferences” on page 98 for more information.
An example QC Report, exportable as a PDF, from a five-laser system is shown.

32 Aurora User’s Guide

Chapter 4: QC & Setup 33
Reference Controls
Reference controls, obtained by running single-stained and unstained samples, provide the
individual fluorescence spectra necessary to unmix the data. Either beads or cells can be stained
for use as reference controls. These controls can be acquired in the reference group of the
experiment during acquisition, or they can be acquired as reference controls in the QC & Setup
module. If reference controls are acquired in the QC & Setup module, they are stored and can be
used as reference controls for unmixing in subsequent experiments.
A wizard guides you through recording reference controls.

Running Reference Controls

To create reference controls you will need to select the fluorescent tags, choose the control type
(beads or cells), then label the fluorescent tags (for example, using CD nomenclature). You can also
append existing FCS files when running reference controls.
Run Daily QC to ensure the instrument is performing optimally before running reference controls.
1 Select Reference Controls from the QC & Setup module.

2 Select New Reference Controls from the Reference Controls tab.

A wizard opens allowing you to create new reference controls. You can also append existing FCS
files.

3 Select the fluorescent tags. The left pane displays the fluorescent tag groups found in the
library.

• Click the arrow to the left of the fluorescent tag group name to view the fluorescent tags in
the group. The default fluorescent tag groups are Blue Laser, Red Laser, Violet Laser, Yellow-
Green Laser, Ultraviolet Laser, Fluorescent Proteins, and Viability Dyes. These contain a list of
commonly used fluorescent tags excited by their respective laser.
• From the expanded list of fluorescent tags, select the fluorescent tags used in the
experiment. Once selected, the fluorescent tags appear in the selection pane on the right.
You can select fluorescent tags by dragging and dropping, double-clicking, or using the Add
button. Multiple tags can be chosen at one time. Confirm the tags selected, then click Next.

34 Aurora User’s Guide

  NOTE: The list of fluorescent tags can be edited in the library. You can use the library to add
fluorescent tags that are not present in the default list. See “Fluorescent Tags” on page 79 for
more information.

4 Define the unstained control(s).

Choose either single-stained fluorescent tags, or new, separate, unstained for the control tubes.
If using a control that has a positive population only, you will need an unstained control of the
same type as the stained.
Either beads or cells can be stained and defined as control types. If you select Use new, separate,
unstained tube(s), you can add more unstained tubes, as necessary. Then select the specific
unstained tube in the Negative Control column of the Fluorescent Tags table below.

5 Define the control type (beads or cells) for each fluorescent tag in the Fluorescent Tags table.

6 (Optional) Enter labels associated with the fluorescent tag for identification and tracking.

7 If applicable, enter the lot number(s) of the reference controls.

Chapter 4: QC & Setup 35

  NOTE: If you selected Label/Lot Specific Unmixing in the Acquisition Preferences (see page 89),
during unmixing, the software will search the library and experiment reference groups for
reference controls that have the same fluorescent tag, label, and lot information to use the
corresponding control for unmixing.

8 Once the controls have been defined, click Next.

9 Load the control sample(s).

• Tube mode: Load the appropriate control sample onto the SIP.
• Loader mode: Assign the location of each control by dragging the control (unstained and
fluorescent tags) to the appropriate location on the plate or tube rack. You can use the auto-
fill feature to fill the remaining wells/tubes. Click Next. Load the carrier by clicking Eject,
loading the carrier on the stage, then click Load to send the stage to the acquisition position.

10 Click Start to preview the sample data. This step allows you to ensure that all populations for all
control samples are on scale. If necessary, adjust gain settings.

36 Aurora User’s Guide

 Gain settings for all channels can be selected from the User Settings drop-down menu, or they
can be individually adjusted for each channel using the detector gain spinboxes (V1–V16, B1–
B14, YG1–YG10, R1–R8, and UV1–UV16).
 NOTE: CytekAssaySetting was generated by measuring the optimal resolution of human
lymphocytes stained with CD8 and CD4 labeled with various fluorochromes. These settings
provide a place to start for most immunophenotyping applications.
The Adjust Settings screen allows you to view the data to ensure that the positively stained
fluorescent particles are on scale. Adjust the threshold and FSC and SSC gains. FSC gain can be
adjusted from 1-1,000. SSC and detector channel gains can be adjusted from 10-10,000. If the
positive population is off-scale for any detector channels, lower the gain setting for that
channel. If the positive population is not sufficiently separated from the negative population
within a specific channel, verify that CytekAssaySetting is selected, as these settings ensure
adequate resolution. If the positive population is still not clearly separated from the negative
population, review the sample preparation and staining procedure.
Once gain settings have been confirmed, unstained and reference controls are ready for
acquisition.
 NOTE: Dim markers may not separate from the negative population regardless of how much
the gain is increased.

11 Preview the data for the remaining controls by clicking Stop, then:
• Tube mode: Loading the next control onto the SIP and clicking Start.
• Plate mode: Selecting the next control well and clicking Start.

12 Select Next when you are satisfied with the gain settings. Proceed to running controls.
13 If you are running in Tube mode, place a tube of the appropriate single-stained particles on the
SIP.

Chapter 4: QC & Setup 37

14 Click Record to begin acquiring.
Make sure to follow the order listed in the left-hand panel. When running in plate mode, you can
select to record a single well/control or the entire plate.
During acquisition the spectra plot for each fluorescent control is displayed. The plots show all
the channels across all lasers in the x-axis vs mean fluorescence intensity (MFI) of the
fluorescent tag.
If the acquisition criteria is not met within 15 minutes, the run will stop.

15 During acquisition, obtain spectral information by moving the polygon gate on the FSC-A vs
SSC-A plot to include the population of interest.

Hold down the Ctrl key while adjusting the gate to move the polygon gates for all the scatter
plots at once. The gated population appears in the histogram, which is set approximately to the
peak emission channel of the fluorescent tag to be acquired. The emission spectrum of the
population is displayed in the spectrum plot.
Adjust the positive gate on the histogram. The software automatically displays the emission
spectrum of the positive particle in the spectrum plot. SpectroFlo software sets the default gate
near or on the peak emission channel. The gate can be selected manually. It is best to set the
gate on the brightest emission, as this can make distinguishing the positive and negative
populations easier and provides better visualization of the spectrum.
 NOTE: Unmixing results are unaffected by the position of the interval gate in the spectrum
plot.
Readjust the positive and/or negative gate on the histogram, if necessary.

16 Continue recording each control.

17 Select Save to save the reference controls.
FCS files for saved reference controls are stored in the export Setup folder by default, or the
folder you set as the default. See “Storage Preferences” on page 97 for information.

38 Aurora User’s Guide

Setting Reference Controls as Benchmarks for Reference Control QC
You can select reference controls to be used as benchmarks for QC. The reference controls you
select as benchmarks allow you to check the quality of your reference controls prior to unmixing. If
a reference control is suboptimal, the benchmark will indicate whether the reference control
spectra appears as expected. If you choose not to designate reference controls as benchmarks, the
reference controls run during QC & Setup or during the experiment will be used as-is without a
quality control check.
 NOTE: Before saving a reference control as a benchmark, ensure that it is fully optimized.
1 Select Reference Controls from the QC & Setup module.

2 Select Benchmark from the Reference Controls tab.

3 Select the reference controls that will be the benchmarks.

Click All to select all the controls in the list.

4 Click Save.

Updating Reference Controls

You may wish to update the reference controls if any of the following occur:
• Major service performed on the instrument
• Fluorochrome exhibiting signs of instability resulting in changes in its emission spectrum
• Staining protocols change, for example different buffers or fixatives are used
The Reference Controls tab displays the reference controls saved in the library. Click the arrow next
to the control name to display the details.

Chapter 4: QC & Setup 39

To Update Reference Controls
1 Select Update Reference Controls from the Reference Controls tab in the QC & Setup module.
A wizard opens allowing you to update reference controls.

2 Follow steps 3 through 12 in “Running Reference Controls” on page 34.

Levey-Jennings Tracking
Levey-Jennings tracks the gain and CV's for all detector channels and the laser delay and area
scaling factor for each laser over time. This allows you to view the system’s performance. Select the
parameter(s) you wish to track.
The graphs in the report show you shifts and trends in the data for each parameter. Data from the
last 30 days, 3 months, or 12 months can be included in the reports.
Select a parameter checkbox to display the Levey-Jennings plot.
Use Save Selection to save the LJ tracking settings.
To export Levey-Jennings data to a .csv file, click Export Data.

40 Aurora User’s Guide

Gain Settings
The amount of signal amplification applied to each detector channel can be modulated by
increasing or decreasing the amount of gain applied. The gains for every detector channel can be
saved and are collectively known as the user settings. User gain settings are stored as a ratio
against the Daily QC. Every time Daily QC is performed, User Settings will be adjusted accordingly.

Alarm Ranges
You can set an alarm to warn you when the gain and %rCV exceeds the passing criteria that you
define. This changes the outliers (shown in red) in the LJ graphs. Select Alarm Range from the
Cytometer QC tab, then adjust the SD range (plus or minus) for individual detectors for each laser.

Chapter 4: QC & Setup 41

42 Aurora User’s Guide
 5
Acquisition

Raw vs Unmixed Data

SpectroFlo software saves flow cytometry data in the FCS 3.1 format. Data is saved in both raw and
unmixed formats. Raw data contains all the fluorescence information from each detector (ie, V1,
V2, V3, etc). Each detector channel is designated by its excitation laser and position in the array. For
example, B3 is the third channel of the blue laser detector array.
Unmixed data contains all the fluorescence information from each fluorescent tag in the
experiment. To unmix data, single stained controls (or reference controls) for each of the
fluorescent tags (as well as an unstained control) are required. During unmixing, an Ordinary Least
Squares (OLS) calculation is used for the decomposition of the fluorescent components in the
sample using the reference controls. Parameters in unmixed data will display as the fluorescent tag
name along with their associated labels.
The Acquisition module provides the tools that allow you to create an experiment. An experiment is
a set of tubes, instrument settings, acquisition criteria (stopping rule), fluorescent tags, labels, and
worksheets designed for the acquisition of samples. See “About Experiments” on page 18.
New and saved experiments can be created or accessed in the Experiments tab of the Acquisition
module.

Unmixing and Compensation

Raw FCS files can be spectrally unmixed in the following ways:
• Reference group from the experiment – Reference controls collected as FCS files within the
experiment can be used to unmix using the Unmixing wizard in the Acquisition module.
• Reference controls run in QC & Setup module – Reference controls run in the QC & Setup
module can be used to unmix using the Unmixing wizard in the Acquisition module.
• Unmixing from the Extra Tools module – FCS files collected from different experiments can be
unmixed in the Extra Tools module. FCS files can be imported and unmixed in this module.
Raw FCS files can also be compensated with the conventional method using the Virtual Filters tab in
the Extra Tools module. Detector channels can mimic the analysis of the data as if it were acquired
using a filter. See “Virtual Filters” on page 74 for more information.

Chapter 5: Acquisition 43
Setting Up an Experiment
An experiment can be saved as a template or created for one time use. Setting up the experiment
in SpectroFlo software involves:
1 (Optional) Providing a name and description for the experiment. A default name is provided.

2 Specifying the fluorescent tags used in the experiment.

3 Defining the reference group with associated reference tags, labels, and lot numbers, as
needed.

4 Adding labels and lot numbers to each of the fluorescent tags.

5 Adding custom keywords. Custom keywords can be defined in the Library.

6 Selecting an acquisition worksheet—either new or a template.

7 Defining acquisition criteria (stopping rule based on events, time, or volume).

Acquisition Experiment Overview

The Acquisition module provides the necessary elements for data collection within the experiment.
Click the Experiment icon in the far left pane to open a template, the default, or a new experiment
using the wizard.

Experiment Display
The experiment display in the Acquisition module includes the following panes. To show, hide, or
undock (float) these panes from the experiment panel, click the corresponding icons in the top-
right corner of the pane.

Group-Tube List and Hierarchy

The samples are listed in the upper left of the screen. Samples can be organized into groups. Use
the Tube and Group icons to add tubes and groups. If running from a Loader, click Add Plate
or Rack (ASL only) to add a new sample carrier. Click Plate View to display a graphic image of the
carrier instead of the list of groups.

44 Aurora User’s Guide

Click Save to save changes to the experiment, or click Save As to save an experiment template. Click
Edit to edit the experiment. Worksheets can be applied to the experiment, groups, or individual
tubes.
Tube mode

Acquisition Control
The Acquisition Control pane allows you to start, stop, choose flow rate, pause acquisition, record
data, and restart acquisition counters. The acquisition controls are enabled when a tube is present
on the SIP or when in plate mode. To show, hide, or undock (float) this pane from the experiment
panel, use the dock/undock and hide icons in the top-right corner.
For information on the Loader Acquisition Controls when in plate mode, see “Loader Acquisition
Controls” on page 112.

The following table describes the controls in the Acquisition Control pane.

No. Control Description

1 Start/Record/Pause/ Start and Record are enabled when a tube is present on the SIP.
Stop/Restart Select Start to start acquisition
Select Record to record data. Record can also start acquisition.
Select Pause to pause recording. While paused you can adjust the
flow rate. Select Record again to continue.
Select Stop to stop acquisition.
Select Restart to restart the acquisition counters. All events and
results displayed are refreshed.
Stop and Restart are enabled once Start is selected.
Stop and Pause are enabled once Record is selected.

Chapter 5: Acquisition 45
 No. Control Description

2 SIT Flush Select to perform a SIT Flush

3 Flow Rate Select Low (15 µL/min), Medium (30 µL/min), or High (60 µL/min).
The exact flow rate is displayed.
4 Event Rate, Abort Displays the real-time counts during acquisition.
Rate, Threshold
Count, Time Elapsed
5 Events to Display Enter the number of events to display during acquisition.

Instrument Control
The Instrument Control pane consists of the Gain, Threshold, Signal, and Lasers tabs for use in
adjusting the instrument.
User Settings allow you to select CytekAssaySetting, Default, or any saved user settings for the
experiment. We recommend using CytekAssaySetting as a starting point. This setting provides the
optimal resolution for each channel, accommodates bright signals, and minimizes spread. While
using CytekAssaySetting, you will need to only adjust FSC, SSC gains, and Threshold.

2
3

46 Aurora User’s Guide

The following table describes the tabs in the Instrument Control pane.

No. Tab Description

1 Gain Gains can be adjusted for all detector channels for all lasers using the gain
spinboxes. FSC gain can be adjusted from 1–1,000. SSC and fluorescence
detector gains can be adjusted from 10–10,000. To change the value that the
gain increments, see “Acquisition Preferences” on page 88. Use All Channels
% to increase/decrease all gains for a selected laser by the percentage you
select.
2 Threshold Use the Threshold tab to set the threshold parameter and minimum
threshold channel value. Multiple parameters can be set as a threshold
using either the AND or OR operator. Use OR when at least one parameter is
available.
3 Signal Use the Signal tab to select area, height, or width for each signal. Area and
height can be selected for all channels. Width can be selected for only one
channel per laser.
4 Lasers Use the Lasers tab to set the area scaling factor and laser delay. These
values are automatically set and updated in all user settings upon
completion of the Daily QC.
 NOTE: If you run large cells and the lowest FSC gain setting is not low
enough to see your cells, lower the FSC area scaling factor (for example, 0.5).

Chapter 5: Acquisition 47
Worksheets
The worksheet allows you to view the data in plots and create plots, statistics, population hierarchy,
and gates. Click the worksheet icon in the far left pane to select, import, save, and print worksheets.

Worksheet Toolbar
A toolbar at the top of the worksheet area allows you to undo/redo, zoom; create plots, gates,
statistics, population hierarchy, annotations; and save, print, and save a PDF of the worksheet.
Hover the cursor over an icon to see a description and keyboard shortcut.

Plots
Four plot types can be created in the worksheet:
• dot plots
• pseudocolor plots (density plots)
• histogram plots
• spectral plots

48 Aurora User’s Guide

To change the properties of a plot, right-click the plot and select Properties. You can select the plot
type, parameters, scale, background color, and labels.

Gates
Gates types include:
• Rectangle
• Ellipse
• Polygon
• Interval
• Quadrants and Hinged Quadrants (select and drag an offset handle to move the quadrant
segment up or down)
• Binary

Click and drag handle to move quadrant

segment up or down.

Chapter 5: Acquisition 49
Gate Properties
Gate properties can be changed by right-clicking on the gate and selecting gate properties. You can
change the name of the gate, the color, and gate boundary line weight. You can also select whether
to display the count and/or the % parent events within the gate, as well as the gate parameters.

Boolean Gates
Select multiple gates in the Population Hierarchy window and right-click to open a menu to:
• Intersect the gates with the AND operator – events that are present in all of the selected gates
are part of the intersected gate population.
• Join the gates with the OR operator – events that are present in at least one of the gates are part
of the joined gate population.

Statistics
To create a statistics box, click the Statistics icon in the worksheet toolbar, then click in the
worksheet area.

Select the population checkbox next to the populations that have stats to display. To add a statistic,
select the statistic from the Statistics Variable list.
Select the parameter you would like to add for the statistics. Multiple parameters can be selected at
once.
The software offers a counts/µL statistic that can be calculated for any gate.
To adjust the precision of the statistics, select the decimal place in the Decimal Places table. To
remove a statistic, right-click the column header and select Delete.

50 Aurora User’s Guide

Once the statistics are complete, you can export the stats as a CSV file to the location you choose.
Statistics output can be appended or overwritten if you export the stats into the same CSV file.

Adjusting Spillover
You can adjust the fluorescence spillover for a selected tube/well.
1 Ensure the green arrow is next to the tube/well and right click, then select Edit Properties in the
group-tube hierarchy list.
The Tube/Well Properties window opens.

2 Select the Spillover tab.

3 Click the Enable Compensation checkbox to activate. The Adjust Spillover icon in the toolbar is
now enabled.

4 Leaving the Tube/Well Properties window open, click the Adjust Spillover icon , then click and
drag in the plot in the direction you want to adjust.

Chapter 5: Acquisition 51
 Changes are reflected in Tube/Well Properties window.

5 Click Save to save the changes. To discard the changes, close the Tube/Well Properties window
and click No in the confirmation window, or click Discard changes in the tube/well properties
window.

Creating a Default Experiment

Use the default experiment option to quickly begin an experiment. The default experiment
contains a set of fluorescent tags, however, all parameters are user configurable.
1 Click Default in the Acquisition Experiment menu.

The default experiment opens with a default raw worksheet.

52 Aurora User’s Guide

2 Click Edit.

The same wizard appears as when creating a new experiment. Follow the steps to select the
fluorescent tags, add groups, and define makers, keywords, acquisition settings/worksheets,
and Loader settings.
See “Creating a New Experiment” on page 53 for details on defining the information in the
experiment wizard.

Creating a New Experiment

Selecting New in the Experiment menu opens the New Experiment wizard. The wizard walks you
through the steps to create a new experiment.
1 Click New in the Acquisition Experiment menu.

Chapter 5: Acquisition 53
2 The Create New Experiment wizard opens. Specify a name for the experiment or use the default
name. (Optional) Type in a description.

3 Click the arrow to the left of the group name (laser) in the Library pane on the left to display the
list of its fluorescent tags. Select the fluorescent tags used in the experiment and click Add to
add them to the Selection list on the right. You can also double-click the tag to add it to the
Selection list.

To quickly find a fluorescent tag, type the tag name in the Type to filter text box. A default list of
fluorescent tags for each group is available in the library. See “Fluorescent Tags” on page 79.
You must select all fluorescent tags present in the experiment, as this will determine which
reference controls are to be used during spectral unmixing.

54 Aurora User’s Guide

 To remove individual fluorescent tags from the Selection list, click to select the tag, then click
Remove. To remove all fluorescent tags, click Clear All.

4 Once all fluorescent tags have been chosen from the Library list, confirm the list in the selection
pane, then click Next.

5 Ensure the correct Carrier Type (Manual Tube) is selected, then create groups for your samples
by selecting Group. Add tubes to the groups.

For information on creating groups when in plate mode, see “Creating Groups When Using the
Loader” on page 114, then proceed to step 7.
 NOTE: In order to minimize carryover, especially if running sticky samples in a plate, we
recommend adding cleaning wells between samples to thoroughly clean the mixing probe. For
example, add two wells, one with 10% bleach and the next with DI water. At the end of a plate,
consider adding a group of four wells, two with 300 µL of 10% bleach and two with 300 µL of DI
water. Program a long mix (15 seconds at 1500 rpm) to thoroughly clean the mixing probe.

6 Select Reference Group if you are intending to unmix with all or some controls acquired in
this experiment.

This creates a list of reference control tubes for each fluorescent tag specified as part of the
experiment.
 NOTE: If you plan to unmix the samples using only reference controls run in QC & Setup,
step 6 is not necessary.
 NOTE: When using controls from the reference library and running controls live, you will
delete the controls to be used from the library and run the other controls. When unmixing, you
will add the controls from the library to unmix all the controls.

Chapter 5: Acquisition 55
7 IMPORTANT: Define an unstained control for autofluorescence by selecting its control type
(beads or cells). The unstained control needs to be of the same type and prepared in the same
way as the samples, as this will ensure accurate unmixing and autofluorescence quantitation.

8 If applicable, select Define Additional Negative Control(s) for Spillover Calculation to use a different
unstained control to calculate spillover for your reference controls. Then enter a name and
control type for this extra negative control.

For example, if test samples are cells and the reference controls are beads, all with only positive
peaks, you will need to run a separate tube of negative beads for the spillover calculation. An
extra negative control is not needed if your unstained autofluorescence control (and sample) is
the same type as the reference controls. If you need to have additional negative controls, see
“Negative/Unstained Controls” on page 63.

9 Select the control type (beads or cells) for the single-stained reference controls.

10 (Optional) Enter the label (for example, CD nomenclature) that is conjugated to the fluorescent
tag.

11 If applicable, enter the lot number(s) of the reference controls.

 NOTE: If you selected Label/Lot Specific Unmixing in the Acquisition Preferences (see page 89),
the software will search the library and experiment reference groups for reference controls that

56 Aurora User’s Guide

 have the same fluorescent tag, label, and lot information in order to use the corresponding
control for unmixing.

 NOTE: Use the red trash can icon to delete an individual tube from the reference group. This
may be necessary if you wish to mix and match references acquired in this experiment with
reference controls run in QC & Setup. Any stored controls you plan to use should be deleted
from the reference group.

12 Click Save.
Once the reference group has been created, entries for each of the references will be displayed.
Each of the reference group tubes/wells will have an icon with the letter R associated with it
under the reference group.

13 If necessary, continue adding tubes/wells, click Next when all tubes are created.
14 Add markers/labels to the remaining sample tubes before continuing. They can be chosen from
the Labels list on the right, typed directly into the table, or copied and pasted. Labels can be
added at the group or tube level and can be applied to multiple cells selected at once. Labels

Chapter 5: Acquisition 57
 are required for reference controls if you selected Label/Lot Specific Unmixing in the Acquisition
Preferences (see page 89). Click Next when all the tubes are labeled.

15 (Optional) Enter custom keywords and click Next.

Custom keywords can be added at the experiment, group, or tube level. You must define the
custom keywords in the Library before you can add them to an experiment (see “Keywords” on
page 81 for information). Drag and drop the keywords from the Keywords list on the right to the
experiment, group, or tube and enter keywords values as needed. You can also copy and paste
custom keywords across different tubes in this wizard.

16 Select the acquisition settings and worksheet(s). The worksheet menu lists all the worksheets
for the given user.

• Select the Default Raw Worksheet (Raw) for the Reference Group and for the sample groups
if you plan to perform post-acquisition unmixing.
• Select the Default Unmixed Worksheet (Unmixed) or any user-created unmixed worksheet
for your sample groups if you are performing live unmixing. Worksheets can be selected at
the experiment, group, or tube level.
Select the stopping gate, storage gate, number of events to record, stopping time (in seconds),
or stopping volume (in µL). Acquisition stops when the first of the stopping criteria is met (time,

58 Aurora User’s Guide

volume, number of events). These criteria can be selected at the experiment, group, or
individual level.
• If acquiring beads, we recommend collecting 5,000 singlet events.
• If acquiring cells, we recommend collecting 10,000 to 20,000 events of the desired
population.

 NOTE: If running in plate mode, when selecting worksheets and acquisition settings, select
the top level (group or experiment) to apply the settings to all wells within the group or
experiment.
 NOTE: The number of events to acquire depends on the target population. For example, you
may need to acquire 10,000 to 20,000 events to get 2,000 of the desired population.
Approximately 1,000 to 2,000 events is needed in both the negative and positive populations of
each control for accurate unmixing.

Select Tube/Well Specific User Settings if you want to apply specific user settings to individual
tubes/well. If left unchecked all tubes/wells in the experiment will use the same experiment
user settings. For more information on user settings, see “User Settings” on page 83.

Chapter 5: Acquisition 59
17 If running in plate mode, define the Loader settings. See “Defining Loader Settings” on
page 116.

18 Once the worksheet and stopping criteria have been defined, click Save and Open to open the
new experiment.

To make any changes to the experiment, click Edit above the group/tube hierarchy.

19 To acquire controls and samples and perform live unmixing, see “Live Unmixing” on page 64.
20 If running in Plate mode, calibrate the SIT. See “Calibrating the SIT” on page 119.

60 Aurora User’s Guide

 6
Unmixing and Compensation

Spectral Unmixing
Spectral unmixing is an important concept to understand how data is generated and analyzed
using the Aurora flow cytometer with SpectroFlo software. Spectral unmixing is used to identify the
fluorescence signal for each fluorophore used in a given experiment.

Understanding Full Spectrum Flow Cytometry

Because fluorophores emit light over a range of wavelengths, optical filters are typically used to
limit the range of frequencies measured by a given detector. However, when two or more
fluorophores are used, the overlap in wavelength ranges often makes it impossible for optical
filters to isolate light from a given fluorophore. As a result, light emitted from one fluorophore
appears in a non-primary detector (a detector intended for another fluorophore). This is referred to
as spillover. In conventional flow cytometry spillover can be corrected by using a mathematical
calculation called compensation. Single-stained controls must be acquired to calculate the amount
of spillover into each of the non-primary detectors.
The Aurora's ability to measure a fluorophore’s full emission spectra allows the system to use a
different method for isolating the desired signal from the unwanted signal. The key to differentiate
the various fluorophores is for those to have distinct patterns or signatures across the full
spectrum. Because the system is looking at the full range of emission of a given fluorophore, and
not only the peak emission, two dyes with similar emission but different spectral signatures can be
distinguished from each other. The mathematical method to differentiate the signals from multiple
fluorophores/dyes is called spectral unmixing and results in an unmixing matrix that is applied to
the data. While not mathematically identical to conventional compensation, the overall principal is
the same. Just as for compensation, single-stained controls, identified in SpectroFlo software as
reference controls, are still necessary, as they provide the full fluorescence spectra information
needed to perform spectral unmixing.
One advantage of unmixing is the ability to extract the autofluorescence of a sample and treat it as
a separate parameter. This is especially useful when running assays with particles that have high
autofluorescence and for which that high background has an impact in the resolution of the
fluorescent signals. Per experiment, you can define one unstained control or multiple unstained
controls (one per group), depending on whether the multicolor samples have the same or different
autofluorescence signatures.

Chapter 6: Unmixing and Compensation 61

Spectrum plots from conventional spectrum viewer
shows heavy overlap between Qdot™ 705 and BV711
peak emission spectra.

Spectrum plots from Aurora show distinct signatures

for Qdot™ 705 and BV711.

Unmixing Workflows

Unmixing Overview
There are three unmixing workflows available in SpectroFlo software—two in the Acquisition
module and one in the Extra Tools module:
• live unmixing during acquisition
• post-acquisition unmixing (in the Acquisition module)
• post-acquisition unmixing (in the Extra Tools module)
When data is acquired with live unmixing, references are acquired as raw data either in the
experiment as part of the reference group or previously acquired in the QC & Setup module as
reference controls. References for all fluorescent tags used in a given experiment must be present
in the system in order for live unmixing of multicolor samples to occur. The live unmixing
functionality allows you to visualize unmixed data during acquisition.

Reference Controls for Unmixing

Depending on when you unmix the data, you will use the following controls for unmixing.

Unmixing Reference Controls

Live unmixing Reference controls run in the experiment or reference controls

run in QC & Setup
Post-acquisition unmixing in Reference controls run in the experiment or reference controls
Acquisition module run in QC & Setup
Post-acquisition unmixing in Any FCS files from samples run in any experiment
Extra Tools module

Multicolor samples can be acquired as raw data and unmixed post acquisition as well. This can be
done in either the Acquisition module or the Extra Tools module.

62 Aurora User’s Guide

 8QPL[LQJ:RUNȵRZV

LIVE UNMIXING POST-ACQUISITION UNMIXING

In Acquisition Module
Acquire all reference controls In Extra Tools Module
(in experiment or from QC & Setup). In Acquisition Module
Acquire all reference controls Reference controls and samples
and samples. were acquired previously (FCS files).
[Reference controls may not have
been run as reference controls, or may
Click Unmix not all be in the same experiment.]
Click Unmix

Click 6SHFWUDO8QPL[LQJ

Check Autofluorescence Extraction

box, if desired; adjust gates in FSC vs
Check Autofluorescence Extraction
SSC, histogram, and spectrum plots.
box, if desired; adjust gates in FSC vs
SSC, histogram, and spectrum plots.

Import FCS files.

Click Live Unmix Parameters must match. Designate
Click &UHDWH1HZ8QPL[HG([SHULPHQW sample types and fluorescent tags for
samples that are single stained.

Adjust gates in FSC vs SSC,

histogram, and spectrum plots.
Acquire samples in an unmixed worksheet.
Both raw and unmixed FCS files are saved.
New Analysis experiment is created
automatically. Only unmixed FCS
Click Unmix
files are saved.
Raw data file are saved in the
original experiment.
Only unmixed FCS files are saved.

Negative/Unstained Controls
In addition to positive reference controls needed for spectral unmixing, an unstained control is also
necessary to assess autofluorescence. The unstained control needs to be of the same type and
prepared in the same way as the samples, as this will ensure accurate unmixing and
autofluorescence extraction, if desired. Ideally, your reference controls, negative control, and
samples will all be the same sample type and prepared in the same way.
In addition to assessing autofluorescence, fluorescence spillover must also be determined. To
correct for spillover, the unstained autofluorescence control can be used if it matches the sample
and reference control type. However, if your reference controls do not match your sample type and
do not contain a negative population in each tube (have only positive peaks), you must use a
separate spillover unstained control that matches your reference control type.
Controls

reference controls (can be beads or cells)

Sample
Cells autofluorescence unstained control (must be cells with same characteristics as samples)

spillover unstained control (can be beads or cells, must match reference controls)

Chapter 6: Unmixing and Compensation 63

Live Unmixing
Samples can be unmixed during acquisition. Live unmixing can be performed with the reference
group acquired during the experiment, the reference controls (run during QC & Setup and stored in
the system), or a combination of both.
For each sample tube that is live unmixed, two FCS files are generated, one that is composed of raw
data and one that is composed of unmixed data.
Live unmixed data can be analyzed in unmixed worksheets in the Acquisition module. Unmixed
worksheets are different from raw worksheets, as they only display fluorescence information
categorized into the defined fluorescent tags for each of the experiments.

To Perform Live Unmixing

1 Create a new experiment with fluorescent tags defined. Create a reference group in the
experiment with the fluorescent tags, if there are any that have not already been stored as
reference controls. See “Creating a New Experiment” on page 53 for details.

2 To view the data for the reference control tubes, make sure CytekAssaySetting is selected, then
click Start. If necessary, use the Instrument Controls to adjust the settings so that all events are
on scale. View all the controls, as well as the multi-color tube, and make any instrument
adjustments to ensure populations are on scale before you begin recording.

To edit the acquisition criteria, click Edit at the experiment level and select the Acquisition tab.
Or, to edit the properties of a single tube, right-click a tube and select Tube Properties.
 NOTE: Keep in mind the more events you acquire, the longer it takes to unmix the data.
3 Click Record when you are ready to begin acquisition. Acquisition stops when the first stopping
criterion is met.

 NOTE: If necessary, you can pause to change the flow rate.

64 Aurora User’s Guide

4 When all reference controls are acquired, click Unmix in the upper-left toolbar.

5 For the negative controls, we recommend selecting Use Control from Experiment if unmixing with
controls you acquired in the experiment.

6 If using reference controls from the Library in QC & Setup, select Use Controls from Library.

Checkmarks appear for those controls coming from QC & Setup. The checkbox is only active if
reference controls for those fluorescent tags are already saved with the reference controls from
the QC & Setup module.

7 Click Next.

8 Use the Identify Positive/Negative Populations tab to include the positive and negative
populations for each fluorescent tag in the appropriate gate.

Only the data plots for the samples you acquired are displayed, not for reference controls that
you chose to use from the library.
 NOTE: If you need to set the FSC and/or SSC axis to a log scale, select the Log checkbox.

a. Move the polygon gate in the FSC vs SSC plot on the left to include the singlet population.
Hold down Ctrl to move all the polygon gates at once.

Chapter 6: Unmixing and Compensation 65

 b. Move the positive interval gate in the histogram to include the positively stained population.
Move the negative interval gate to include the negative population if not using a separate
negative control.
c. Move the interval gate on the spectrum plot on the right to select the channel that exhibits
the brightest fluorescence intensity. This channel is the peak emission channel for the
fluorescent tag.
 NOTE: If one of the controls is questionable or does not contain sufficient data, you can
reacquire it or append to it, then unmix again.

9 (Optional) To see how the reference controls run in the experiment compare to the benchmark
reference controls, click Next.

 NOTE: For information on creating benchmarks, see “Setting Reference Controls as

Benchmarks for Reference Control QC” on page 39.
Two options allow you to view how the two reference controls compare—spectral profile and
Similarity™ Index.
• Spectral Profile displays the emission spectrum of the unmixing controls against benchmark
spectra designated by the user. The benchmark reference control spectra appear in red and
the reference controls appear in black.
A Similarity Index appears to the right of the plots. A value closer to 1 indicates similar
spectral signatures, while a value closer to 0 indicates spectral signatures that are not similar.
For comparison to benchmarks, the value should be close to 1. If the value is below 0.97, it
will be flagged with a yellow warning symbol. This indicates a mismatch of the unmixing
control spectra with the benchmark spectra. If the Similarity Index falls below this value, it is
imperative to check the unmixing control against the reference spectra provided in the
fluorochrome guideline found in the Help menu. See “Similarity Matrix” in the following
section.
The Similarity Index can also be used when viewing and comparing the full spectral
signatures of any two dyes. Dyes with similar spectral signatures can be challenging to
resolve. In this case, it is best to use dyes with a Similarity Index 0.98. See
spectrum.cytekbio.com for a full spectrum viewer tool.

66 Aurora User’s Guide

 If no benchmark control is established for a particular dye, that plot will only display a black
line that represents the spectrum of the unmixing control. The Similarity Index will display N/A.

• Similarity Matrix displays a Similarity Index matrix and a Complexity™ Index value.
Click View Similarity Index above the matrix to display the indices for each dye. The Similarity
Matrix will display the Similarity Index for each dye against itself and all the other dyes to be
unmixed in the experiment.
The Complexity Index is a measure of how distinguishable a collection of spectral signatures
are from each other when unmixed together. It calculates this by looking at the ratio of the
Similarity Index of the worst overlapping combination of signatures to the best overlapping
combination of signatures.

Chapter 6: Unmixing and Compensation 67

10 Click Live Unmix.

11 The wizard closes and the experiment reappears. The reference group now has the unmixed
icon to the left of the tube(s). Select an unmixed worksheet to view the unmixed data.

12 Select the sample tube you wish to acquire. The green arrow indicates the tube is selected.
Click Start, then Record.

Use My Experiments to open experiments you ran if you wish to review the data or acquire
more samples.

FCS files are stored in the Export folder by default, or the folder you set as the default. See
“Storage Preferences” on page 97 for information. FCS files for live unmixed data are saved as
both raw data and unmixed data.

Analyzing Data Offline

To analyze data offline, you can click My Experiments, select the experiments you want to export,
right-click and select Export. This will export the entire experiment as a ZIP file with all of the FCS
files and worksheet templates contained inside. This experiment can be imported into other
instances of SpectroFlo software, or unzipped to access the FCS files for analysis using other
analysis software.

68 Aurora User’s Guide

Post-Acquisition Unmixing
Samples can be acquired as raw data and then unmixed after acquisition is complete. This can be
done through two methods:
• post-acquisition unmixing in the Acquisition module (see below)
• post-acquisition unmixing in the Extra Tools module (see page 70)

Post-Acquisition Unmixing in the Acquisition Module

The unmixing wizard in the Acquisition module limits reference controls to those coming from the
reference group in the experiment or reference controls run in QC & Setup.
To perform post-acquisition unmixing in the Acquisition module, perform the same workflow as
live unmixing.
 NOTE: Steps 1 and 2 are the same as live unmixing. Step 3 is used to create a new unmixed
experiment.
1 Acquire all reference control tubes and sample tubes prior to selecting the Unmix button in the
upper-left pane.

2 Examine the spectral plots by doing the following, if needed:

a. Move the polygon gate in the FSC vs SSC plot on the left to include the singlet population.
Hold down Ctrl to move all the polygon gates at once.
b. Move the positive interval gate in the histogram to include the positively stained population.
Move the negative interval gate to include the negative population.
c. Move the interval gate on the spectrum plot on the right to select the channel that exhibits
the brightest fluorescence intensity. This channel is the peak emission channel for the
fluorescent tag.

3 Click Create New Unmixed Experiment.

Chapter 6: Unmixing and Compensation 69

 A new experiment opens with a new unmixed worksheet.

Post Acquisition Unmixing in the Extra Tools Module

When performing post-acquisition unmixing in the Extra Tools module, you can pick and choose
which FCS files to unmix (for example, controls coming from different experiments, reference
controls run during QC & Setup, or single-stained controls that were not run as part of the
reference group).
FCS files can be designated into three categories:
• Single Stained
• Unstained
• Sample
 NOTE: There must be at least one single-stained FCS file and one unstained FCS file in the file list.
Otherwise, unmixing cannot be performed.
 NOTE: The parameters need to match in order for this to work.
In addition, raw FCS files can also be conventionally compensated in this module through the
Virtual Filters tab. This function can simulate the presence of filters and can compensate data using
conventional compensation methods (see “Virtual Filters” on page 74).

To Unmix Raw Data Files:

1 Select Spectral Unmixing from the Extra Tools module.

2 Click Import to import raw FCS files for unmixing.

3 Select the files. Select multiple files using either the Shift or Ctrl key. Click Open.

70 Aurora User’s Guide

4 Upon importing, a dialog box on how to assign sample types appears. Read the instructions
and click OK.

5 Once FCS files have been imported, select the sample type for each FCS file as Single Stained,
Unstained, or Sample. The software will automatically designate the type based upon the file
name. You can manually modify these if the automatic designation is incorrect.

6 FCS files designated as single-stained will require a fluorescent tag designation to specify what
reference spectrum will be provided for unmixing.

Chapter 6: Unmixing and Compensation 71

7 Enter a label for each single-stained control and sample.

8 Select Universal Negative for single-stained FCS files that do not contain a negative population,
and the unstained control will be used for the negative population. In the bottom left of the
screen, check whether Auto Fluorescence will be used as a fluorescent tag.

9 Click Show Plots to display the data in the FSC vs SSC plot, peak emission channel histogram,
and spectrum plots.

10 The positive and negative populations need to be identified through the appropriate placement
of the existing gates. Click OK to adjust the gates.

a. Move the polygon gate in the FSC vs SSC plot to include the singlet population.
b. Move the interval gate in the histogram labeled Positive to include the positively stained
population. Move the interval gate in the histogram labeled Negative to include the negative
population. Do not adjust the negative gate when using the Universal Negative.

72 Aurora User’s Guide

 c. Move the interval gate on the spectrum plot on the right to select the channel that exhibits
the brightest fluorescence intensity. This channel is the peak emission channel for the
fluorescent tag.

11 Click Unmix.
12 Select the directory to which the unmixed FCS files are exported or leave the default. Click OK.

These FCS files can then be imported to an experiment for analysis or analyzed using third-
party software.

Chapter 6: Unmixing and Compensation 73

Virtual Filters
The Virtual Filters option in the Extra Tools module allows you to compensate raw FCS data using
conventional compensation methods.
1 Click the Virtual Filters tab in the Extra Tools module.

2 Click Import to import raw FCS files for virtual filter analysis.

These FCS files can be single-stained reference controls, unstained controls, and/or sample files.
However, you must include an unstained control FCS file.

3 Upon importing, a dialog box on how to assign sample types appears. Read the instructions
and click OK.

4 Once FCS files have been imported, the sample type for each FCS file needs to be designated as
Single Stained, Unstained, or Sample. The software will automatically designate the type based
upon the file name. You can manually modify these if the automatic designation is incorrect.

5 FCS files designated as single stained require a fluorescent tag designation. Select the
fluorescent tag for each single-stained sample.

If there is no negative population in the single-stained FCS file(s), select Universal Negative, and
the unstained control will be used for the negative population.

74 Aurora User’s Guide

The virtual filter is automatically assigned by the software based upon the fluorescent tag
designation. (Optional) To increase the bandwidth of the virtual filter, use the channel pull-
down menus to select the desired range. See the following table for wavelength ranges.

The following table shows the system’s filter bandwidths.

Center Bandwidth Wavelength Wavelength End

Laser Channel
Wavelength (nm) (nm) Start (nm) (nm)
UV1 373 15 365 380
UV2 388 15 380 395
UV3 428 15 420 435
UV4 443 15 436 451
UV5 458 15 451 466
UV6 473 15 466 481
UV7 514 28 500 528
UV8 542 28 528 556
Ultraviolet
UV9 582 31 566 597
UV10 613 31 597 628
UV11 664 27 651 678
UV12 692 28 678 706
UV13 720 29 706 735
UV14 750 30 735 765
UV15 780 30 765 795
UV16 812 34 795 829

V1 428 15 420 435

V2 443 15 436 451
V3 458 15 451 466
V4 473 15 466 481
V5 508 20 498 518
V6 525 17 516 533
V7 542 17 533 550
V8 581 19 571 590
Violet
V9 598 20 588 608
V10 615 20 605 625
V11 664 27 651 678
V12 692 28 678 706
V13 720 29 706 735
V14 750 30 735 765
V15 780 30 765 795
V16 812 34 795 829

Chapter 6: Unmixing and Compensation 75

 Center Bandwidth Wavelength Wavelength End
Laser Channel
Wavelength (nm) (nm) Start (nm) (nm)
B1 508 20 498 518
B2 525 17 516 533
B3 542 17 533 550
B4 581 19 571 590
B5 598 20 588 608
B6 615 20 605 625
B7 661 17 653 670
Blue
B8 679 18 670 688
B9 697 19 688 707
B10 717 20 707 727
B11 738 21 728 749
B12 760 23 749 772
B13 783 23 772 795
B14 812 34 795 829

YG1 577 20 567 587

YG2 598 20 588 608
YG3 615 20 605 625
YG4 661 17 653 670
Yellow- YG5 679 18 670 688
Green YG6 697 19 688 707
YG7 720 29 706 735
YG8 750 30 735 765
YG9 780 30 765 795
YG10 812 34 795 829

R1 661 17 653 670

R2 679 18 670 688
R3 697 19 688 707
R4 717 20 707 727
Red
R5 738 21 728 749
R6 760 23 749 772
R7 783 23 772 795
R8 812 34 795 829

6 (Optional) Select a label for the single-stained controls and samples.

76 Aurora User’s Guide

7 Click Show Plots to display the plots.

The data is displayed in the FSC vs SSC plot and fluorescent tag histogram plot.

8 The positive and negative populations need to be identified through the appropriate placement
of the gates. Click OK to adjust the gates.

a. Move the polygon gate in the FSC vs SSC plot to include the singlet population. Hold down
Ctrl to move all the polygon gates at once.
b. Move the interval gate in the histogram labeled Positive to include the positively stained
population. Move the interval gate in the histogram labeled Negative to include the negative
population. Do not adjust the negative gate when using the Universal Negative.
The histogram x-axes are labeled with the fluorescent tag instead of the channel/detector.

Chapter 6: Unmixing and Compensation 77

9 Click Calculate Comp once gates have been set correctly. The conventionally compensated data
is displayed. To view the data for a specific FCS files, select the file.

The spillover matrix is also calculated. Click Spillover Matrix to view the spillover values.

10 Click Export and select the location where you wish to export the conventionally compensated
data. The files are exported to a folder named Compensated followed by the current date and
time. Files can then be imported back into an experiment to analyze in SpectroFlo software.

78 Aurora User’s Guide

 7
Library, Preferences, and Users

Library
The library contains information for various elements used for the experiments. Information saved
in the library includes SpectroFlo QC bead lots, fluorescent tags, labels, user settings, worksheet
templates, experiment templates, keywords, and Loader settings. Information stored in the library
can be saved, exported, and imported for reuse.

QC Beads
SpectroFlo QC bead lot IDs and expiration dates can be imported, exported, or removed from the
library. The QC bead lot for the beads used for Daily QC must be imported into the library. Select
QC Beads to see a list of QC bead lots.

Fluorescent Tags
Fluorescent tags are the designation given to each distinct fluorescent molecule that can be
detected by the system. This includes for example, fluorophores, fluorescent proteins, and
fluorescent viability dyes. Each unique fluorophore run on the instrument must be given a
fluorescent tag name.
By default, several groups of fluorescent tags are pre-installed with the software—Blue Laser, Red
Laser, Violet Laser, Yellow-Green Laser, Ultraviolet Laser, Fluorescent Proteins, and Viability dyes.

Chapter 7: Library, Preferences, and Users 79

These groups contain the most commonly used fluorophores excited by the system’s on-board
lasers. Additional tags can be added to these groups.

You can create groups or individual fluorescent tags by selecting Add. Both groups and individual
fluorescent tags can be imported or exported. Note: Selecting New will create a new group while
selecting Add will allow you to add an individual fluorescent tag to a group.
To edit the properties of a fluorescent tag that you added, select the fluorescent tag of interest and
select Edit. Properties that can be edited include fluorescent tag name, laser excitation wavelength,
emission wavelength, and display name. The default tags that are included with the software
cannot be edited or deleted. However, they can be exported and imported across different systems
as long as you are using the same version of SpectroFlo software.
If the fluorophore is known by another name or identified by a different spelling, those additional
names or spellings can be added as synonyms.
Any groups that you created can also be edited in this window. The default groups cannot be edited
or deleted.

Labels
Fluorescent tags can be conjugated or attached to proteins that can specifically bind to other
proteins on the cell surface or within the cytoplasm. They can also be inherently fluorescent, such as
fluorescent proteins that can be fused to a variety of cellular proteins using molecular cloning
techniques. The proteins that are either bound or attached to fluorescent tags can be designated as
labels. The software comes with an initial set of pre-installed labels that are categorized as CD

80 Aurora User’s Guide

Markers, Chemokines, Chemokine Receptors, and Cytokines. Additional labels can be added by using
Add in the right pane.

New label groups can be created by clicking New. Label groups can also be imported and exported
for use on other systems. The default groups and labels cannot be edited or deleted.

Keywords
Keywords allows you to type, import, or export user-defined, custom FCS keywords by group. These
keywords can be assigned in an experiment at the experiment, group, or tube level and are
exported with the FCS files.
The following types of keywords can be created:
• Numeric
• String
• Boolean
• Selectable numeric

Chapter 7: Library, Preferences, and Users 81

• Selectable string

Standard Keywords
The following table describes the standard FCS 3.1 keywords found in SpectroFlo FCS files.

Keyword Description

$FIL Tube or well name

$PLATENAME Plate name
$WELLID Well ID
$VOL Total sample volume (microliters)
$DATE Date that the FCS data was acquired
$BTIM Beginning time of data acquisition
$ETIM End time of data acquisition
$OP Operator name
$INST Institution name
$CYT Name of cytometer (Aurora)
$CYTSN Cytometer serial number
$SPILLOVER Spillover matrix

Custom Keywords
The following table describes the custom keywords found in SpectroFlo FCS files. Custom keywords
are not required as part of the FCS 3.1 standard.

Keyword Description

APPLY COMPENSATION True or False to indicate whether compensation is enabled when FCS
file is loaded into SpectroFlo
CHARSET Encoding. UTF8.
CREATOR Software name and version
FSC ASF FSC area scaling factor
GROUPNAME Plate name

82 Aurora User’s Guide

 Keyword Description

LASER{x}DELAY Laser delay for each laser

LASER{x}ASF Area scaling factor for each laser
LASER{x}NAME Name for each laser
P{x}DISPLAY Preferred display scale
PLATEROWS Number of rows of the plate
PLATECOLS Number of columns of the plate
THRESHOLD Threshold channel and value
TUBENAME Name of sample tube
WINDOW EXTENSION Window extension used to record the tube/well
USERSETTINGNAME The user setting name used to record the tube/well

You can change the value of a custom keyword, then select Auto Update for that keyword to update
the keyword in subsequent experiments.

Spillovers
Spillovers shows the spillover matrices saved during experiments. These saved matrices can be
used for other experiments. For more information on the spillover matrix, see “Adjusting Spillover”
on page 51.

User Settings
User Settings are the set of gain settings, threshold, and signal type for all detector channels. The
date when it was created and modified, as well as the name of the user who created it are also

Chapter 7: Library, Preferences, and Users 83

saved. The settings are saved from the Instrument Control Pane in the Acquisition module. The
name and description can be modified in this tab.
Select a user(s) to view the user settings created by that user(s). Click the pen tool to edit the
description of the selected user settings. Administrators can check the Shared checkbox to allow all
users to see and use the settings created by other users. SuperUsers can view, import, and export
user settings, but cannot deleted settings created by other users.
To delete user settings that are no longer needed, select it, then click Delete.

Loader Settings
Loader settings can be saved and are stored in the library. The settings name, date the settings
were created, version, as well as the name of user who created them are displayed. Click Description
to add information about the settings.
Loader settings include mix time and speed, stage temperature setting, sample return, number of
SIT flushes, and record data delay time.
For more information on Loader settings, see “Loader Settings” on page 113.

Worksheet Templates
All worksheets created in the Acquisition module are saved in the library and can be accessed
through the Worksheet Templates tab. Worksheets can be exported as WTML files and imported
for re-use. For more information on worksheets, see “About Worksheets” on page 21.

84 Aurora User’s Guide

Select a user(s) to view the worksheet templates created by that user(s). Click the pen tool to edit
the description of the selected worksheet. Administrators can check the Shared checkbox to allow
all users to see and use the worksheet templates created by other users. SuperUsers can view,
import, and export worksheet templates, but cannot delete templates created by other users.
To view a worksheet, select it, then click View. To delete a worksheet that is no longer needed, select
it, then click Delete. The default worksheets cannot be deleted. You can also set the default raw and
unmixed worksheets back to their original defaults by selecting the worksheet, then clicking Restore
Default.

Experiment Templates
Experiment templates contain the key elements of an experiment without the data. They can be
saved and stored in the library for future use. The name, creation date and time, description, and
creator information is displayed. Experiment templates can also be imported and exported from
this tab. For more information on experiments, see “About Experiments” on page 18.
Select a user(s) to view the experiment templates created by that user(s). Click the pen tool to edit
the description of the selected worksheet. Administrators can check the Shared checkbox to allow
all users to see and use the experiment templates created by other users. SuperUsers can view,
import, and export experiment templates, but cannot delete templates created by other users.

Chapter 7: Library, Preferences, and Users 85

To delete an experiment template that is no longer needed, select it, then click Delete. The default
experiment template cannot be deleted.

Backup & Restore

Users and SuperUsers can back up selected data from their accounts directly from SpectroFlo
software. This includes experiments, experiment templates, worksheets, user settings, Loader
settings, daily QC results, and reference controls. Administrators have the additional ability to back
up selected data from the different User and SuperUser accounts.
1 To back up data, click Backup in the right pane.

2 Choose a timeframe (day, week, month, or custom timeframe) that includes the data, select the
user(s).

3 Select the data type you want to back up, and click Backup.

86 Aurora User’s Guide

 To restore these settings, click Restore and browse to the location where the settings are saved.

Preferences
The Preferences module allows you to change various functionality and display elements of the
software user interface. Each user has the ability to change their own preferences. The following
sections describe the options that can be changed in the Preferences module. Each section within
the Preferences can be restored to its default settings by selecting Restore Default Preferences.

Chapter 7: Library, Preferences, and Users 87

Acquisition Preferences
In the Acquisition tab, you can change the number of events displayed on plots during acquisition,
the number of SIT flushes, the preview time before recording begins, and the value that the gain
settings increment when you click the spin-box arrow during acquisition. You can also enter default
prefixes for tube, group, plate, tube rack, and experiment names. A label/lot specific unmixing
feature allows you to choose specific reference controls for unmixing. The Batch Analysis options
allow you to choose the output file type and tube/well results contained in the PDF.

The following table describes the options in the Acquisition preferences.

Item Description

Number of Events to The number of events displayed in the pseudocolor plots, dot
Display on Plots plots, and histograms. The default is 2,000 events.
Number of Automatic SIT Choose the number (0–2) of SIT Flushes that will be performed
Flush After Tube Unload once a tube is removed from the SIP.

88 Aurora User’s Guide

Item Description

Data Review Time (Sec) The number of seconds that elapse before the tube pointer moves
After Tube Recorded to the next tube after the current tube is finished recording.
Record Data Delay Time The number of seconds before it begins recording after you click
(Sec) for Manual Tube Record.
Gain Spinbox Up/Down Increments the gain for each detector channel by the amount
Increment (Ctrl key Held) indicated when you hold the Ctrl key and select the up and down
arrows of the Gain spinbox.
Experiment Enter the default name for tubes, groups, plates, tube racks (ASL
only), and experiments. Edit the default name and/or append to
the existing names.
Select the default user settings for experiments.
Ref Controls Ordered by Control Type allows you to order
reference controls by type (beads or cells). If unchecked, reference
controls are ordered by excitation laser, then by peak emission.
Unmixing Feature Label/Lot Specific Unmixing allows you to use lot-specific
reference controls for unmixing when appropriate. If this option is
selected, the software will search the library and experiment
reference groups for reference controls that have the same
fluorochrome, label, and lot information in order to use the
corresponding control for unmixing.
Select the preferred SSC channel for unmixing (SSC for the signal
from the violet laser (default), or SSC-B for the signal from the blue
laser).
Batch Analysis Select the default output file type for batch analysis reports (PDF,
CSV, or both).
Choose to create a single PDF for all tubes/wells, or individual PDF
files for each tube/well.

Chapter 7: Library, Preferences, and Users 89

Worksheet Preferences
The Worksheet tab allows you to change the way elements are displayed in the worksheet. Header
and footer properties are also adjusted in the Worksheet tab.

The following table describes the options in the Worksheet preferences.

Item Description

Population Hierarchy Sets the default height and width for the population hierarchy
Window Size experiment element.
Statistics Table Window Sets the default height and width for the statistics table.
Size
Grid Display Grid – Toggles on/off the display of grid lines in the worksheet.
Display Page Line – Toggles on/off the page break line in the worksheet.
Grid Size – Modifies the size of the grid squares. Options include 1”,
1/2”, 1/4”, and 1/8”.
Snap to Grid – Toggles on/off the ability for the worksheet elements to
snap to and line up with the grid lines on the worksheet.

90 Aurora User’s Guide

 Item Description

Page Setup Title – Select a worksheet title from a list of default titles. The title is
shown when the worksheet is printed or exported as a PDF.
Show & Print Page Number – Toggles whether the page number is
shown and printed.
Print Header & Footer – Toggles whether the header and footer are
printed. Allows you to select the text that is displayed in the left and
right headers and footers.
Print Grid – Toggles whether the grid is printed. Can also be set to use
Page Setting.
Page Layout – Toggles between landscape and portrait.
Page Size – Sets the page size according to standard paper sizes.
Page Margin – Sets the margins of the page to Narrow, Normal, or Wide.

Plot Preferences
The display properties of pseudocolor, dot, and histogram plots can be adjusted in the Plot tab.

Chapter 7: Library, Preferences, and Users 91

The following table describes the options in the Plot preferences.

Item Description

Default 2D Plot Size Set the default height and width of the pseudocolor plots and dot
plots in pixels.
Default Histogram Size Set the default height and width of histograms in pixels.
Default Spectrum Size Set the default height and width of spectrum plots in pixels.
Default Background Color Set the default background color for all plots. Click to select from the
available colors.
Default Plot Title Customize the title of all plots to include the plate name, group
name, tube name, population name, and/or a custom name.
Pseudocolor Plot Density Increase or decrease the number of density levels displayed in the
Levels pseudocolor plot.
Histogram Smooth Set whether histogram distributions are smoothed.
Histogram Filled Set whether histogram distributions are filled.
Histogram Y Axis Set the scale of the histogram y-axis to count or percentage.
Image Export Format Select the format (PNG, TIFF, or JPEG) and resolution (96, 200, 300, or
500 dpi) for exported images.

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Gates Preferences
Gate properties can be adjusted in the Gates tab.

The following table describes the options in the Gates preferences.

Item Description

Default Name Location Select where the gate name is displayed with respect to the gate
itself.
Show % of Parent Toggles on/off the display of the % of Parent with the gate name.
together with Gate Name
Show Count together Toggles on/off the display of the population count with the gate
with Gate Name name.
Gate Boundary Line Sets the thickness of the line drawn by the gate.
Weight
Default Colors for First X Set the number of gates (1–10) that will follow the color scheme
Gates detailed in the gate color table. The order in which the colors appear
can be changed.
Interval Gate Default Toggles on/off whether the population captured by the interval gate
Color has a default color.

Chapter 7: Library, Preferences, and Users 93

 Item Description

Quadrant Gate Default Select whether the population captured by the quadrant gate has a
Color default color, and if so if each quadrant has a different color.
Binary Gate Default Color Select whether the population captured by the binary gate has a
default color.

Statistics Preferences
The default degree of precision (number of decimal places) of the statistics displayed in the
worksheet can be modified in the Statistics tab.
The precision for the following statistics can be adjusted: Mean, rSD, % rCV, Mean, Max, Min, SD,
% CV, % Total, % Parent, % Grand Parent, % Specified Gate, Count/µL, and Absolute Count.

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Fonts Preferences
The Fonts tab allows you to change the font properties of each display element.

The following table describes the options in the Fonts preferences.

Item Description

Font Locations Select which display element's font to modify.

Text Settings Font Family – Select the font family.
Font Size – Select the font size.
Color – Select the font color.
Font Style – Toggles between normal and italic.
Font Weight – Select normal, bold, or semibold.
Sample Text Shows a preview of the text with the properties set in the Text Settings.

Chapter 7: Library, Preferences, and Users 95

Annotation Preferences
You can adjust the text properties for annotation text.
Select the font family, size, style, weight, and alignment, as well as the foreground, background, and
border colors. Selected text options appear under Sample Text.

Notifications Preferences
The Notifications tab allows you to change certain notification settings in the Acquisition and Extra
Tools modules.

96 Aurora User’s Guide

The following table describes the options in the Notifications preferences.

Item Description

Acquisition Module Toggles whether to display the Save Changes pop-up window when
closing an Experiment, Worksheet, and User Settings, as well as
whether to display a confirmation message when canceling the
experiment wizard.
Extra Tools Module Toggles whether to display the instructional dialog boxes in the Extra
Tools module.
Window Sizes and Select Restore Default to set pop-up window sizes and locations to
Locations the defaults.

Storage Preferences
The Storage tab allows administrators to set the default storage locations for the experiment FCS
files and setup FCS files. This option is available only for administrators.
 NOTE: Do not move files stored in these folders.

The following table describes the options in the Storage preferences.

Item Description

FCS Files Folder Select the folder where FCS files are saved.
FCS files from experiments where only raw data was acquired, as well
as FCS files from experiments where live unmixing was performed are
stored here. For experiments where live unmixing is performed, both
raw and unmixed FCS files are saved.
• You can select to label FCS files with group names as the prefix. Note
that you can set the default group name in Acquisition preferences.
• You can select to organize FCS file folders by date. If not selected,
FCS files are ordered by experiment name, without a parent folder
indicating the date.
If you change the default folder where FCS files are saved and want to
move existing experiments to the specified folder, click Migrate Now.

Chapter 7: Library, Preferences, and Users 97

 Item Description

Setup FCS Files Folder Select the folder where FCS files generated by QC & Setup procedures
are saved.

QC Setup Preferences
The QC Setup tab allows you to select the days/months of QC reports to display in the Reports
section of the Cytometer QC menu in the QC & Setup module.

Cytometer Preferences
The Cytometer tab allows you to select the default carrier type (manual tube, 96-well plate, 96-well
deep-well plate, or tube rack) and select how bubbles and clogs are handled by the system. These
selections are for systems configured with a loader. Carrier configuration settings allow you to set
SIT lift distance for tubes and plates and select a throughput option for plates.

98 Aurora User’s Guide

The following table describes the options in the Acquisition preferences.

Item Description

Default Carrier Type Choose the carrier type for sample delivery method – Manual Tube
for manual sample delivery, or the appropriate 96-well plate, 96-
well deep-well plate, or tube-rack if you are using a Loader.

Chapter 7: Library, Preferences, and Users 99

 Item Description

Fluidics Skip Fluidics Boost when Starting Acquisition – select this feature if
you wish to skip the boost that quickly delivers sample from a
tube/well to the flow cell at the start of acquisition.
NOTE: The Skip Fluidics Boost feature works only for Medium and
High flow rates.
Enable Bubble/Clog Detection – allows you to choose how you
want to proceed in the event of a bubble or a clog when running a
plate. You can either move to the next well or stop immediately
after a SIT flush.
• If two consecutive clogs are detected when acquiring from a
plate, acquisition stops immediately, regardless of how the
Bubble/Clog Detection option is set.
• Disabling SIT Flush (see Number of Automatic SIT Flush After Tube
Unplugged in Acquisition preferences) also disables the SIT flush
step during clog detection. For proper functionality, ensure the
automatic SIT Flush is enabled when Bubble/Clog Detection is
enabled.
Cytometer Warm Up Select to enable data acquisition during the recommended
30-minute instrument warm period. This feature can only be
enabled by an administrator.
Sample Carrier Select (enable) the sample carrier – If using a Loader, you must
Configuration have Manual Tube and at least one other carrier type selected.
SIT Lift Distance – The distance in millimeters from the tip of the
SIT to the bottom of the well or tube.
Default Loader Setting – Choose the default, high-throughput, or
low carryover setting when using a Loader.

Users Preferences
The Users tab allows you to select if and when (daily, weekly, monthly) to automatically export the
login sessions. The auto-exported login session files are exported to C:\CytekbioExport\LoginSessions.
For more information on exporting login sessions, see “Use Time” on page 103.

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Global Information
Enter information for your department in the Global Information tab.

Users
User accounts can be managed in the Users module. User account information and use time are
stored in the module.
There are three types of user accounts (roles)—Administrator, Super User, and User. Only
Administrators can manage user accounts.

Managing Users
Administrators can add, delete, edit, and disable user accounts from the Users tab. User passwords
can also be changed/reset. The Users tab lists all users and displays the role and status of each.

Chapter 7: Library, Preferences, and Users 101

Adding a New User Account
1 Click Add New in the User tab of the Users module.
This option is available only for administrators.

2 Enter a user account ID and password, then enter the password again to confirm.

 NOTE: The user account ID appears in the user name filed in the User’s tab.
3 (Optional) Enter the user’s full name, email, and phone number.

4 Select the user role—Administrator, SuperUser, or User.

5 Select the account status—enabled or disabled.

6 Click Save.

Editing a User Account

1 Select the user from the User tab of the Users module, then click Edit.

2 You can edit or add a user name, email, and/or phone number. You can also change the user
role and account status.

3 Click Save.

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Resetting a User Password
An administrator can change their or another administrator’s password and reset an operator’s
password.
1 Select the user from the User tab of the Users module.

2 Click Reset Password.

3 Click Yes to confirm resetting the password.

4 A dialog opens displaying the new password.

5 Make note of the password and click OK.

Deleting a User Account

To delete a user account, select the user from the User tab of the Users module, then click Delete.
When you remove a user account all the account information and that data acquired by that user is
deleted. The FCS files saved by the user are retained.

Use Time
Use Time shows the total daily, weekly, and monthly use time (duration) and number of sessions
that each user is on the system. Select a user(s) to view and export the use times for the selected
user account(s). Click on the checkbox next to the user in the Select User(s) section to be able to
view the sessions for use time to export. Select the session or all the sessions for export.

To see a comprehensive list of every login session for selected user(s), select All. The log on (start)
and log off (end) times for each session, as well as the session duration are displayed.
Administrators can manage the list of login sessions by deleting all sessions before a specified

Chapter 7: Library, Preferences, and Users 103

date. To delete sessions, select the user(s) or click User to select all users, click Manage Use Time,
select the date, and click Delete. Manage Use Time appears when you select All.

Administrators can export daily, weekly, monthly, or all use times and login sessions to .csv files by
selecting the user(s) and session dates and clicking Export Selected, or selecting user(s) and clicking
Export All to export all use times and login sessions.

User Role
Administrators can edit the privileges for User and SuperUser user roles. Administrators can also
create and edit new custom user roles. Administrator privileges cannot be changed.

Editing a User Role

1 Select User Role from the Users tab, then select the user name (role) you wish to edit.
The default user roles (and names) are Administrator, SuperUser, and User. The Administrator
role cannot be edited.

2 Click Edit to edit the selected user role.

3 Edit the user role privileges, then click Save.

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 See “User Role Privileges” in the following section.

4 If you wish to restore user role privileges to the defaults, click Restore Default in the Edit User
Role window.

User Role Privileges

The following table shows the privileges for the three default user types (Administrator, SuperUser,
and User). Administrators can edit the privileges for the SuperUser and User. Administrator
privileges cannot be edited.

Privileges User SuperUser Administrator

Instrument Control
Change fluorescence gains  
Customize parameters (signal types)  
Window extension  
Area scaling factors   
Change laser delay  
Enable data acquisition during warm up  
(cytometer preference)
Library Documents
Manage QC bead lot  
Create fluorescent tags   
Create label   
Create reference controls   

Chapter 7: Library, Preferences, and Users 105

 Privileges User SuperUser Administrator

User Management
Manage users  
Manage all user types 
Create user role 

Creating a User Role

Administrators can create new user roles based on the privileges of the three default roles.
1 Select User Role from the Users tab.

2 Click New.

3 Enter a name for the new user role.

4 Select the base type for new role. Base types are Administrator, SuperUser, and User.

The new role can have the same privileges as the selected base type. You can remove some
privileges but you cannot grant more privileges than the base type has. For example, if you
want the new user role to manage QC bead lot information, select SuperUser as the base type,
as the User base type does not have this privilege.

5 (Optional) Enter a description.

6 Click Save.

The new user role appears in the Name list.

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Deleting a User Role
Administrators can delete a custom user role.
1 Select User Role from the Users tab.

2 Click Delete.

3 Click Delete to confirm deletion.

User Policy
Administrators can configure general password and login security policies.
1 Select User Policy from the Users tab to display the user policies.

2 Select the following:

• if and when passwords expire

• if and when passwords can be reused
• password strength
– strong is 8–20 characters with upper-case and lower-case letters, at least one number, and
at least one special character
– medium is 6–20 characters with letters and numbers
– weak has no restrictions
• if and when a maximum number of login attempts fail (users are locked out of SprectroFlo
after the selected number of unsuccessful login attempts is reached)
• if and when users are automatically logged out if inactive for the selected time (1 minute to
45 hours, in various increments)
• change default password when login next time (if the default password Rainbow is set, the
user will be prompted to create a new password)
• enable login username typing prompt (as you begin to type when logging in, a list of user
names with matching characters appears)

Chapter 7: Library, Preferences, and Users 107

108 Aurora User’s Guide
 8
Loader

Loader Overview
The Automatic Micro-Sampling System (AMS) and the new Automated Sample Loader (ASL), both
referred to as Loader throughout this guide, are optional sample loading accessories that add high-
throughput sample acquisition capabilities to the Aurora.
The AMS resuspends samples via a mixing probe, which vortexes each well individually. It supports
a variety of 96-well plates. The Loader plate stage houses a metal element that can cool/heat from
4°–30°C, if you choose. A wash station, located at the back of the plate stage is used to clean the
mixing probe.

wash station

The new ASL resuspends samples using an orbital shaker. In addition to supporting a variety of
96-well plates, including deep-well plates, it supports a 40-tube rack.
Loader settings are user-adjustable, including mixing speed, mixing duration, the number of SIT
flushes between samples, sample recovery, and data record delay time. Pre-defined Loader
settings include the default settings for high throughput mode, standard mode, and low carryover
mode.
When the Loader is powered on and successfully connected to the software, the status indicator in
the lower-right corner of the screen shows a green checkmark. A red X appears if the Loader is not
powered on or not connected to the software.

Chapter 8: Loader 109

Using the Loader
The Loader must be calibrated for the plate type you will be using. For information on calibrating
the Loader for the plate, see “Calibrating a Plate” on page 118.

Enabling the Loader

1 Turn on the power to the Loader. The power switch is at the back of the Loader where the fluidic
lines connect.

2 If there is a tube on the SIP, remove it.

3 When running 96-well plates, pull the lever towards you. On the AMS, this brings forward the
mixer. On the ASL, this brings forward the guide. Use this on the ASL to run all plates, 96 deep
well and standard well.

When running 40-tube racks on the ASL, this lever should be left in the back position.

power switch

4 Before running your experiment, you must perform a SIT calibration to set the proper SIT depth
within the well. See “Calibrating the SIT” on page 119.

Loading a High-Throughput Sample Carrier

1 If necessary, click Eject from either the QC & Setup or Acquisition module to eject the stage,
bringing it forward.

2 Load a plate or tube rack on the stage so that position A1 is located in the front-left corner. Push
the plate against the back of the plate stage, then press down on the front edge of the plate to
secure it with the clips in the holder.

3 Click Load from the QC & Setup or Acquisition module to load the plate, followed by Start to
begin acquisition.

4 To run tubes, select Manual Tube as the carrier type option in the experiment.

Minimum Well Volume

When acquiring from a well, a boost is applied to deliver the sample to the flow cell as quickly as
possible. This process results in the loss (consumption) of up to 35 µL of sample, as the sample is

110 Aurora User’s Guide

neither acquired nor recorded. We recommend a minimum volume of 50 µL of sample per well to
accommodate for this.
 NOTE: The amount of sample consumed as lost can be reduced further by selecting to skip the
fluidics boost. If the boost is disabled, it will take longer for the sample to reach the flow cell. You
will need to take into account the acquisition delay for recording FCS files. There is a chance that no
adjustment to the acquisition delay could result in not all desired events being recorded. For
information, see “Acquisition Preferences” on page 88.

Loader Settings in the Experiment

This section lists the differences in the Acquisition module when using a Loader. The following
panes are described:
• Group Hierarchy (“Group Hierarchy/Plate Display” on page 111)
• Acquisition Controls (“Loader Acquisition Controls” on page 112)
• Loader Settings (“Loader Settings” on page 113)

Group Hierarchy/Plate Display

The Group and Tube hierarchy pane when using a Loader defaults to one group with one tube. It
also provides an option to add a plate. You can view either a hierarchical list (List View), or once a
plate is added, a graphical representation of the plate (Plate View).

Default Experiment List View Default Experiment Plate View Once Plate is Added

Chapter 8: Loader 111

Loader Acquisition Controls
The Acquisition Control pane allows you to start, stop, and pause acquisition, record data, and
restart acquisition counters. To show, hide, or undock (float) this pane from the experiment panel,
use the dock/undock and hide icons in the top-right corner.

The following table describes the controls in the Acquisition Control pane.

No. Control Description

1 Start/Record/Stop/ - For AMS: Start and Record are enabled when the Loader is on, the
Load/Eject/Restart lever is pulled forward, and the Sample Delivery Mode in
Acquisition preferences is set to Plate.
- For ASL: It will depend on whether running a plate or running a
tube rack. Running a plate: the lever is forward. Running a tube
rack: the lever is to the back.

Select Start after selecting Load plate to start acquisition.

Select Record to record data. Record can also start acquisition.
Select Stop to stop acquisition.
Select Load or Eject to load or eject the plate.
Select Restart to restart the acquisition counters. All events and
results displayed are refreshed.
Stop and Restart are enabled once Start is selected.
Stop and Pause are enabled once Record is selected.
2 SIT Flush Select to perform a SIT Flush
3 Flow Rate Select Low (15 µL/min), Medium (30 µL/min), or High (100 µL/min).
The exact flow rate is displayed.
4 Event Rate, Abort Displays the real-time counts during acquisition.
Rate, Threshold
Count, Time Elapsed
5 Events to Display Enter the number of events to display during acquisition.

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Loader Settings
Several default Loader settings are available. You can select the Default, High Throughput, or Low
Carryover Loader Setting, depending on your application. You can also create custom settings.
Loader settings are enabled once a plate is added. Adjust the settings before acquisition.
AMS ASL

The following table describes the AMS and ASL Loader settings.
AMS:
ASL:
Both

Setting Description

Selected Settings Three Loader settings are available—default, high throughput, and low
carryover. You can create your own custom settings.
Acquisition order Select the order that you want the plate to run.
Wells are acquired by:
• row from left to right (A1-A12, B1-B12, etc)
• column from top to bottom (1A-1H, 2A-2H, etc)
• row from left to right, then right to left (A1-A12, B12-B1, C1-C12, etc)
• column from top to bottom, then bottom to top (1A-1H, 2H-2A, etc)
Mix Time Select time (in seconds) that each well is mixed. You can also disable
the mix time.
Mix Speed Select the speed at which the mixer spins (in RPM).
Shake Time Select the time (in seconds) to shake the plate/tube rack. You can also
disable shake time.
Shake Speed Select the speed of the orbital shaker (in RPM)
Shake Interval Mode Select whether you would like to shake every N wells or after a specified
period of time. You can also disable shaking.

Chapter 8: Loader 113

 Setting Description

Shake Every N Wells, Select how often (number of wells or time in seconds) to shake the
or Shake Interval plate/tube rack.
Premix Time Select the time (in seconds) to shake the plate/tube rack before
acquisition of the first tube/well.
SIT Flush Times A SIT flush is performed over the wash station after each acquisition.
Choose single flush, double flush, or disabled if you do not wish to
perform a SIT flush.
Sample Recovery Allows any remaining sample that is left in the SIT after acquisition is
complete to be deposited back into the well.
Stage Temperature Select the plate stage temperature (4°–30°C).
Record Data Delay Select the time in seconds you wish to preview data from a well before
Time recording begins once you click Record.

Experiments in Plate Mode

Follow the instructions “Creating a New Experiment” on page 53 to create a new experiment. The
steps to create a new experiment when running in plate mode are the same as when running in
Tube mode, except for the following tasks—creating groups and defining Loader settings—outlined
below.

Creating Groups When Using the Loader

 NOTE: When running sticky samples, we recommend adding cleaning wells between samples to
thoroughly clean the mixing probe. For example, add two wells, one with 10% bleach and the other
with DI water. At the end of a plate, consider adding a group of four wells, two with 300 µL of 10%
bleach and two with 300 µL of DI water. Program a long mix (15 seconds at 1500 rpm) to
thoroughly clean the mixing probe.
Before you can add groups when creating a new experiment, you must add a plate.
1 To add groups when running in plate mode, ensure the correct Carrier Type is selected, then click
Add Plate.

114 Aurora User’s Guide

 A plate image appears on the right. Three icons at the far right allow you to add groups.

2 Click in the plate image to select a well, or click and drag to select multiple wells corresponding
to the wells in the group you wish to add, then click the appropriate icon to the right of the plate
to define the sample types in the group:

• To add a group for samples, click +.

• To add a group for reference controls, click R.
If you are intending to unmix with controls acquired in this experiment you must add a
reference group. When you add a reference group you will be prompted to define the
reference controls. See step 7 on page 56 for details on defining the unstained and
fluorescence controls.
• To add a group for cleaning well(s), click C. For example, you can add DI water to wells to
rinse the SIT and clean the mixing probe to prevent carryover.
Wells will be marked as sample (S), reference control (R), or cleaning (C).

Chapter 8: Loader 115

3 When all groups have been defined, click Next.

4 Follow steps 14 and 16 starting on page 57 to continue creating the experiment.

Defining Loader Settings

1 The last step when creating an experiment is to select the Loader settings. Loader settings can be
applied to all wells in the plate or at the group level. The settings for the two Loader types differ
slightly. See “Loader Settings” on page 113 for information.
We recommend starting with Default settings for most immunophenotyping applications.
 NOTE: Default settings were optimized using PBMC's and human whole blood.

116 Aurora User’s Guide

2 Once the worksheet and stopping criteria have been defined, click Save and Open to open the
new experiment.

To make any changes to the experiment, click Edit above the group/well hierarchy.

3 To acquire controls and samples and perform live unmixing, see “Live Unmixing” on page 64.

Chapter 8: Loader 117

Minimum and Maximum Volumes for Carrier Types

Carrier Type Min Volume (µL) Max Volume (µL)

96-well standard well plate, round bottom 50 200

96-well standard well plate, U-bottom 50 200
96-well standard well plate, V-bottom 50 200
1 mL 96-well deep well plate, round bottom 50 500
2 mL 96-well deep well plate, round bottom 50 1000
40-tube rack, 12 x 75 mm tubes 50 2000

Calibrating a Plate
The plate calibration feature allows administrators to calibrate the stage for each of the compatible
96-well plates. Use the controls to align the SIT within well A1 and adjust the depth of the mixer in
the well. The settings are saved automatically. Use the plate calibration feature if you change plate
types.
For a list of compatible plates, see “Supported Carrier Types” on page 142.
1 Click the Plate Calibration icon in the Acquisition module.
The Plate Calibration option is available only to administrators.

118 Aurora User’s Guide

 The plate calibration screen appears.

2 Load the plate that you wish to calibrate.

3 Select the plate type from the drop-down list.

4 Select A1 Position to calibrate the X and Y directions.

• Use the Move Stage buttons to align the SIT within the well.
• Click Reset if you wish to revert to the default settings.

5 Select the Mix Motor Depth to calibrate the Z direction.

• Ensure the SIT is positioned above well A1 and click Continue.

• Use the Move Mixer Down button to move the mixing probe deeper into the well. Use the
Move Mixer Up button to move the mixing probe to a shallower position.
• Click Reset if you wish to revert to the default settings.

 NOTE: A Reset All button reverts all settings to the defaults.

The settings are saved automatically and should not need to be changed unless you change the
plate type.

Calibrating the SIT

Use the Calibrate SIT feature to set the proper SIT depth within a plate well. You will need to
perform a SIT calibration:
• prior to running your first experiment of the day in plate mode
• every time you switch from tube mode to plate mode
• if you change plate types
The calibration value applies only to your individual user account. Once calibration is complete, the
settings apply to all plates of the same type.

Chapter 8: Loader 119

 NOTE: A SIT calibration is performed automatically in the tube that is loaded on the SIP when
the system is turned on.
1 Load a 96-well plate.

2 In the Cytometer tab, from either the QC & Setup or Acquisition module, select Calibrate SIT.

3 A dialog appears prompting you to enter the SIT Lift Distance. Enter the distance, then click
Calibrate SIT. The lift distance is the distance in millimeters from the tip of the SIT to bottom of
the well.

The recommended starting point for the SIT Lift Distance is 1.5 for U-bottom plates. Set the
value higher for V-bottom plates. If you experience clogs, try setting the SIT LIFT Distance
higher. For information on changing the default SIT Lift Distance see “Acquisition Preferences”
on page 88.

4 Check the depth of the sample line in the well. If it’s satisfactory, click OK. If it needs to be raised
or lowered, adjust the SIT Lift Distance and click Calibrate SIT.

5 Repeat step 4 until the sample line depth is satisfactory. Click OK.

6 Once SIT calibration is complete, you can run your experiment.

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 9
Maintenance

Maintenance Schedule
Any instrument surface in contact with biological specimens can transmit potentially fatal
disease. Use universal precautions when cleaning the instrument or replacing parts.
Wear PPE such as protective gloves, eyewear, and lab coat.

The 10% bleach solution used throughout the maintenance procedures is prepared by adding
1 part household bleach to 9 parts DI water.
Routine maintenance of the Aurora cytometer includes periodic replacement of parts. For part
numbers, see “Supplies and Replacement Parts” on page 143.

Scheduled Maintenance
The following table describes the scheduled maintenance procedures for your cytometer.

Maintenance Procedure Description Frequency

Replace sheath filter Ensures debris-free sheath fluid Every 6 months, or as needed
Long Clean Cleans the fluidic lines with 10% Once a month and prior to
bleach solution service calls

Unscheduled Maintenance
The following table describes the unscheduled maintenance procedures for your cytometer.

Maintenance Procedure Description Frequency

SIT Flush Backflushes the SIT As needed, if SIT clogs or

after running sticky dyes
Purge Filter Removes bubbles from the sheath If bubbles are present in the
filter sheath filter, or if the plenum
or sheath tank runs dry
Clean Flow Cell Runs 10% bleach solution followed As needed, or after running
by DI water through the flow cell sticky dyes
Clean external surfaces Keeps surfaces free from salt As needed
buildup

Chapter 9: Maintenance 121

SIT Flush
A sample line backflush is performed whenever a tube is removed from the SIP after sample
acquisition. If the sample line exhibits signs of carryover or becomes clogged after completing an
experiment with a sticky dye such as propidium iodide, acridine orange, or thiazole orange, the
sample line should be manually backflushed.
If you are using a Loader, a SIT flush occurs over the wash well, located at the back of the Loader
plate stage.
1 In the Cytometer tab, from either the QC & Setup or Acquisition module, select SIT Flush.

2 If carryover or a clog persists, place a tube of 10% bleach on the SIP and acquire at High flow
rate for 5 minutes. Afterwards, acquire a tube of DI water at High flow rate for 5 minutes.

 NOTE: If running large quantities of beads or large cells, we recommend running a tube of 10%
bleach followed by a tube of DI water, each for 5 minutes, between experiments.

Purge Filter
Perform this procedure if air bubbles are visible in the sheath filter, or if the plenum or
sheath tank have run dry and air is present in the fluidics system.

1 In the Cytometer tab, from either the QC & Setup or Acquisition module, select Purge Filter.

The vent valve connected to the sheath filter will open releasing any air bubbles trapped inside
the sheath filter.

2 Repeat the Purge Filter fluidic mode until there are no visible bubbles inside the sheath filter.

122 Aurora User’s Guide

Clean Flow Cell
Clean the flow cell after completing an experiment with a sticky dye such as propidium iodide,
acridine orange, or thiazole orange. Cleaning the flow cell is also recommended after acquiring
large quantities of highly concentrated bead solutions or if you suspect a clog.
If running sticky samples, perform this procedure using a 30% Contrad 70 solution instead of
10% bleach between experiments.
1 In the Cytometer tab, from either the QC & Setup or Acquisition module, select Clean Flow Cell.

2 Follow the instructions that appear. Load a tube containing 3 mL of 10% bleach solution on the
SIP and click Continue.

3 When prompted, load a tube containing 3 mL of DI water on the SIP and click Continue.

4 Click Done when the procedure is complete.

Long Clean
Decontaminate the fluidics system monthly by running the Long Clean fluidics mode. Run the Long
Clean just prior to service calls, or if you run high volumes of unwashed samples or samples
stained with propidium iodide, acridine orange, or thiazole orange.

Do not run bleach or detergent through the sheath filter. It is difficult to remove cleaning
solutions from the sheath filter.

1 In the Cytometer tab, from either the QC & Setup or Acquisition module, select Long Clean.

Chapter 9: Maintenance 123

2 Follow the instructions that appear. Prepare the appropriate cleaning tubes and fluidics tanks.

3 Empty the waste tank. Replace the sheath filter with the sheath filter bypass (long clean tubing)
assembly.

4 Detach the sheath tank and replace it with a tank containing a 10% bleach solution.

5 Load a tube containing 3 mL of a 10% bleach solution on the SIP.

6 Proceed with the Long Clean in the software.

7 Once the bleach cleaning cycle is complete, reattach the sheath tank.

8 Remove the tube of 10% bleach from the SIP and replace with a tube of 3 mL of DI water.

9 Proceed with the Long Clean in the software.

10 When prompted, remove the long clean tubing assembly and re-install the sheath filter.

Fluidics Shutdown
Perform fluidic shutdown at the end of each day that you use the instrument. The shutdown
procedure thoroughly cleans the fluidics system. If you are running in Tube Mode without a Loader
see “Fluidics Shutdown Using Individual Tubes” in the following section. If you have an ASL, see
“Fluidics Shutdown Using a Tube Rack (ASL only)” on page 125.
To prepare a 10% bleach solution add 1 part household bleach to 9 parts DI water.

124 Aurora User’s Guide

Fluidics Shutdown Using Individual Tubes
1 In the Cytometer menu from either the QC & Setup or Acquisition module, select Fluidics Shutdown.

2 Load a tube containing 3 mL of 10% bleach on the SIP and click Continue.

3 Load a tube containing 3 mL of DI water and click Continue.

4 Load a tube containing 3 mL of 30% Contrad 70 and click Continue.

5 Load a tube containing 3 mL of DI water and click Continue.

6 Allow the shutdown procedure to complete, then click Done and turn off the cytometer. Make
sure the SIT is submerged in the DI water at the end of the procedure.

The following day when you turn on the system, the startup procedure begins, using the tube of
DI water on the SIP.

Fluidics Shutdown Using a Tube Rack (ASL only)

1 In the Cytometer menu from either the QC & Setup or Acquisition module, select Fluidics
Shutdown.

2 Select Tube Rack from the Fluidics Shutdown screen. Prepare and load the appropriate tubes in
the tube rack as instructed.

• Load a tube containing 3 mL of 10% bleach in position A1.

Chapter 9: Maintenance 125

 • Load a tube containing 3 mL of DI water in position B1.
• Load a tube containing 3 mL of 30% Contrad 70 in position C1.
• Load a tube containing 3 mL of DI water in position D1.

3 Click Continue.

4 Allow the shutdown procedure to complete, then click Done and turn off the cytometer.

5 The next time you turn on the system, you are prompted to load a tube of DI water on the SIP.
This is required for the startup procedure to successfully complete.

Cleaning the External Surfaces

Periodically check for saline residue.
1 Dampen a cloth with a mild cleaning solution and wipe the surfaces of the instrument.

2 Dampen a cloth with DI water and wipe the surfaces again to remove residual cleaning
solution.

3 Dry the surfaces with a clean, dry cloth.

Inspecting the Fluidics Lines

Check the cytometer periodically for fluid leaks. If any evidence of a leak is detected,
contact Cytek Technical Support immediately. Do not attempt to repair the instrument.

1 Visually inspect for fluid leaks by looking for small pools of liquid near any of the quick-connects.

2 Visually inspect for dried residue or slight discoloration in the spaces surrounding the
cytometer.

Replacing the Sheath Filter

The sheath filter traps debris and air bubbles before they reach the flow cell. Replace the filter
assembly every 6 months, or when you see increased sample flow rates or debris in an FSC vs SSC
plot.

Wear PPE such as protective gloves, eyewear, and lab coat while performing this
procedure.

Do not run bleach or detergent through the sheath filter, as these fluids can damage the
filter.

1 Turn off the cytometer.

2 Open the front cytometer panel.

126 Aurora User’s Guide

3 Press the two fluidics line quick-connects and the vent line quick-connect to the right of the
sheath filter.

fluidics line quick-connects

vent line quick-connect

sheath filter

4 Discard the sheath filter according to standard laboratory protocol and local regulations.

5 Install a new sheath filter with the arrow pointing up.

6 Restart the cytometer and run the Purge Filter fluidic mode to remove air bubbles (see “Purge
Filter” on page 122). Repeat this step until all air bubbles are purged from the filter.

7 Close the front panel.

Replacing the SIT

Replace the SIT if the tubing is clogged even after repeatedly cleaning and flushing the SIT.
1 Ensure the SIT is extended in a tube of DI water and the cytometer is turned off.
If the cytometer was shut down properly using the Fluidics Shutdown procedure, the SIT will
already be extended and left in water.
 NOTE: If the SIT is not extended, turn on the cytometer and run Fluidics Shutdown. Then
turn off the cytometer.

2 Obtain a SIT tubing assembly.

green ferrule
beige nut black plastic nut

3 Open the SIT door. Identify the three components shown in the following figure.

Chapter 9: Maintenance 127

 Open the front panel, then open the SIT door. The SIT door is located above the SIP. See “Front
of Cytometer” on page 12.

flow meter

black plastic nut

upper beige plastic nut

4 Twist off and carefully remove the black plastic nut from the bottom of the flow meter.

5 Follow the tubing from the black nut down to the beige plastic nut. Twist off the beige nut and
gently pull it and tubing out from the SIP.

6 Discard the SIT tubing assembly.

128 Aurora User’s Guide

7 Insert the new sample tubing through the hole and feed the tubing all the way in.

8 Once the line is in, screw in the beige nut until finger tight. You will feel once the nut is secured
to the bottom.

9 Verify that the tubing contacts the bottom of the tube on the SIP.

Chapter 9: Maintenance 129

10 Secure the black nut to the bottom of the flow meter so it is firmly attached. Make sure the
sample line bends away from the SIT assembly when the SIT stage rises.

Secure black nut.

Ensure sample line bends away from SIT assembly.

11 Close the SIT door.

12 Turn on the system. During initialization the system automatically calibrates the SIT depth in
the tube loaded on the SIP.

130 Aurora User’s Guide

 10
Troubleshooting

This section provides tips to help you identify and resolve issues that might occur on your flow
cytometer. If additional assistance is required, contact Cytek Biosciences. Please have the following
information available: serial number, error messages, and details of recent performance.
For instrument support within the US, call 1-877-92-CYTEK. Visit our website, www.cytekbio.com,
for up-to-date contact information.

Observations

Observation Possible Causes Recommended Solutions

Daily QC does not Wrong QC bead sample Ensure you are running SpectroFlo QC
complete beads.
Bead sample not properly Mix the bead sample.
mixed
Bead sample too dilute Concentrate the bead sample or prepare
a fresh bead sample.
Air bubble in sample line Run a SIT Flush.
Degraded beads Prepare fresh beads.
Warm-up not done Let the instrument warm up for at least
30 minutes.
Daily QC failed Air bubble in fluidics Run a Purge Filter.
Dirty flow cell Run a Clean Flow Cell.
If the problem persists, run a Clean Flow
Cell using 25%–50% Contrad 70, followed
by DI water.
Questionable sample prep Verify the sample prep technique.
Air in sheath filter Run a Purge Filter.
Sample not diluted in same Dilute the sample in the same fluid as the
fluid as sheath sheath solution.

Chapter 10: Troubleshooting 131

 Observation Possible Causes Recommended Solutions

Air in sheath filter Cytometer was not in use for a Run a Purge Filter.
prolonged period Check that all sheath connectors are
securely attached.
Check for leaks or cracks in the sheath
plenum. Replace, if needed.
Empty sheath tank Fill the sheath tank. Run a Purge Filter.
No events displayed No sample in tube Add sample or install a new sample tube.
(flow rate lower
than expected) Sample not properly mixed Mix the sample to suspend cells/particles.
Clogged SIT Run a SIT Flush.
Then run a Clean Flow Cell with 10%
bleach, followed by a Clean Flow Cell with
DI water.
If the clog persists, replace the sample
line.
For loaders, the SIT Lift Increase the SIT Lift Distance. See
Distance set too low (touching “Calibrating the SIT” on page 119.
bottom of tube)
No events displayed Insufficient gain for threshold Increase the gain for the threshold
(flow rate normal) parameter parameter.
Threshold too high Lower the threshold.
Laser delay not correct Ensure the laser delay values match those
from the latest Daily QC run. See
“Instrument Control” on page 46 for the
laser delay location. If the values do not
match, rerun Daily QC.
Threshold set to incorrect Set the threshold to the appropriate
parameter parameter for the application (usually
FSC).
Gated plot with no data in gate Delete or move the gate.

132 Aurora User’s Guide

Observation Possible Causes Recommended Solutions

Low sample event Threshold too high Lower the threshold.

rate
Insufficient gain for threshold Increase the gain for the threshold
parameter.
Sample not properly mixed Mix the sample to suspend cells/particles.
Sample too dilute Concentrate the sample.
Set the flow rate to Medium or High.
Clogged SIT Run a SIT Flush.
Then run a Clean Flow Cell with 10%
bleach, followed by a Clean Flow Cell with
DI water.
If the clog persists, replace the sample
line.
Erratic event rate Partially blocked SIT Run a SIT Flush.
Then run a Clean Flow Cell with 10%
bleach, followed by a Clean Flow Cell with
DI water.
Clumpy sample Vortex, filter, or disaggregate the sample.
Data in scatter Air bubble in flow cell Run a SIT Flush.
parameters appear
distorted Air in sheath filter Run a Purge Filter.
Dirty flow cell Run a Clean Flow Cell.
Poor sample health Check the viability of the cells.
Hypertonic buffers Check the pH of the buffers and fixative.
Incorrect instrument settings Optimize the instrument settings.
High CVs Air bubble in fluidics Run a SIT Flush and a Purge Filter.
Sample flow rate set to High Set the sample flow rate to Low or
Medium.
Dirty flow cell Run a Clean Flow Cell.
If the problem persists, run a Clean Flow
Cell using 25%–50% Contrad 70, followed
by DI water.
Questionable sample prep Verify the sample prep technique.
Air in sheath filter Run a Purge Filter.
Sample not diluted in same Dilute the sample in the same fluid as the
fluid as sheath sheath solution.
SIT hitting bottom SIT Lift Distance set too low Set the SIT Lift Distance to at least 1.5. See
of well/tube “Calibrating the SIT” on page 119.

Chapter 10: Troubleshooting 133

134 Aurora User’s Guide
 11
Glossary

APD Avalanche photodiode (APD) is a highly sensitive semiconductor

electronic device for measuring light intensity. APDs convert light to
electricity and can be thought of as photodetectors.

autofluorescence The inherent fluorescence arising primarily from cell structures such as
mitochondria and lysosomes. Autofluorescence can hinder detection
of dim fluorescent signals.

compensation The process by which spillover fluorescence from secondary

parameters is accounted for so that fluorescence values for a
parameter represent only the fluorescence of the primary fluorophore.

data file A collection of measured values from a single sample combined with
text describing the data that has been stored as a flow cytometry
standard (.fcs) file to disk.

deconvolve An algorithm-based process used to reverse the effects of convolution

(or overlapping) on recorded data.

detector A device that responds to a specific stimulus. Photodiodes and APDs

tubes are two types of detectors in cytometers. They convert light
signals into electronic signals.

dot plot A graphical representation of two-parameter data. Each axis of a plot

displays values of one parameter.

electronic noise Random fluctuation in electronic signals, a characteristic of all

electronic circuits.

event rate The rate at which cells or particles are acquired.

FCS Flow cytometry standard, a standard format for flow cytometry data
files.

Chapter 11: Glossary 135

 filter An optical device that blocks the passage of part of the incident light,
allowing the rest to pass virtually unchanged.

flow cell The flow cell enables hydrodynamic focusing of the sample so that the
individual cells or particles of interest can be interrogated by the
laser(s) sequentially.

flow cytometry A technology that simultaneously measures and analyzes multiple

characteristics of single cells or particles as they pass through a laser
beam.

flow rate The amount of fluid passing through a point per unit of time. The
Aurora’s flow rate is measured by the flow meter.

fluorescence The emission of light of longer wavelengths that occurs when a

substance absorbs light of shorter wavelengths.

fluorophore A fluorescent dye. A molecule capable of absorbing light energy, then

emitting light at a longer wavelength (fluorescence) as it releases this
energy.

gain Amplification of a signal. Increasing gain results in a larger output

signal for a given input signal.

gate A numerical or graphical boundary (region) that defines a subset of

data. Gates can be single- or multi-dimensional.

laser Light Amplification by Stimulated Emission of Radiation. A light source

that is highly directional, monochromatic, coherent, and bright. The
emitted light is in one or more narrow spectral bands, and with most
lasers, is concentrated in an intense, narrow beam.

laser delay Amount of time between signals from different laser intercepts.

photodiode A device for measuring light intensity. A photodiode generates an

output current proportional to the incident light intensity.

rSD Robust standard deviation. The robust SD is based on the deviation of

individual data points to the median of the population.

reference Spectral profile of a fluorescent tag in all detectors for all lasers.

136 Aurora User’s Guide

resolution A measure of a cytometer's ability to distinguish between two
populations with differing fluorescence or light scatter intensities.

sensitivity A measure of a cytometer’s ability to distinguish particles from

background noise. It is often expressed in terms of a minimum number
of fluorescent molecules per particle required to distinguish a stained
particle from an unstained particle. Sensitivity depends on the
instrument, the dye, and the preparation method.

SIP Sample injection port. The area of the cytometer where the sample is
placed.

SIT Sample injection tube. The probe that pulls sample from the sample
tube to the flow cell.

spectral overlap The phenomenon of different fluorophores emitting light within the
same detection range. In multi-color experiments, compensation must
be performed to correct for spectral overlap.

spectral unmixing The mathematical method of deconvolving signals from multiple

fluorophores/dyes to differentiate them.

spillover Emitted light from a fluorophore entering the detector of another

fluorophore.

Chapter 11: Glossary 137

138 Aurora User’s Guide
 12
Specifications

Cytometer

Optics

Item Specification

Optical platform Fixed optical assembly configured with up to five spatially separated
laser beams. Laser delays are automatically adjusted during
instrument QC.
Lasers 355 nm: 20 mW (ultraviolet)
(up to five lasers are 405 nm: 100 mW (violet)
available) 488 nm: 50 mW (blue)
561 nm: 50 mW (yellow-green)
640 nm: 80 mW (red)
NOTE: Ultraviolet and yellow-green lasers are available for Aurora
cytometers only. These lasers are not available for Northern Lights
standard configurations.
Beam geometry Flat-top laser beam profile with narrow vertical beam height
optimized for small particle detection
Emission collection Fused silica cuvette coupled to high NA lens for optimum collection
efficiency to optical fibers
Forward scatter detector High-performance semiconductor detector with 488 nm bandpass
and filter filter
Side scatter detectors Two high-performance semiconductor detectors with 405 nm and
and filters 488 nm bandpass filters
Fluorescence detectors High-sensitivity Coarse Wavelength Division Multiplexing (CWDM),
16-channel semiconductor detector array per laser, enabling more
efficient spectrum capture for dyes emitting in the 420 nm to 830 nm
range. No filter changes required for any fluorophore excited by the
on-board lasers.
NOTE: See the table on page 75 for the detector bandwidth
specifications.

Chapter 12: Specifications 139

Fluidics

Item Specification

General operation Vacuum driven fluidics with the following fluidics modes: Long Clean,
SIT Flush, Purge Filter, Clean Flow Cell, Fluidics Shutdown
Compatible tubes 12 x 75-mm polystyrene and polypropylene tubes
Fluidic reservoirs 4-L fluid tanks with level-sensing provided. Compatible with 20-L
sheath and waste cubitainers.
Sample flow rates Three preset flow rates measured by the flow meter:
Tube mode:
• Low: 15 µL/min
• Medium: 30 µL/min
• High: 60 µL/min
Plate mode:
• Low: 15 µL/min
• Medium: 30 µL/min
• High: 100 µL/min
Data acquisition rate Up to 35,000 events/s (for three-laser systems)

Fluorescence and Scatter Sensitivity

Item Specification

Fluorescence linearity FITC R2 0.995 / PE R2 0.995

Forward and side scatter Enables separation of fixed platelets from noise.
sensitivity
Forward and side scatter Performance is optimized for resolving lymphocytes, monocytes, and
resolution granulocytes.
Side scatter resolution Capable of resolving 0.2-µm beads from noise.

140 Aurora User’s Guide

Workstation

Item Specification

Operating system Microsoft® Windows® 10 Pro, 64-bit

Processor Intel® Core™ i7 processor, 3.6 GHz
RAM 32 GB
Hard drive 500 GB SSD/1TB SATA
Video processor 530 NVIDIA® GeForce
Monitor 32-in UHD 4K Monitor
Minimum requirements Windows 10 OS, Intel Core i7 processor, 16 GB RAM, and 1 TB hard
for SpectroFlo software drive

Installation Requirements

Item Specification

Dimensions (W x D x H) 54 x 52 x 52 cm (21.3 x 20.5 x 20.5 in)

58.4 x 62 x 52 cm (23.0 x 24.4 x 20.5 in) with the Loader
Weight 61 kg (134.5 lb) to 71 kg (156.5 lb)
Workstation 29.1 x 9.25 x 34.4 cm (11.5 x 3.6 x 13.5 in)
Recommended workspace 165 x 76 x 132 cm (65 x 30x 52 in)
Power For the five-laser system:
3.4 A at 120 VAC
1.7 A at 240 VAC
Fuse rating 250 VAC, 5 A, size 5 x 20-mm
Heat dissipation 500 W with all solid-state lasers
Temperature 15°–28°C
Environmental conditions Indoor use
Altitude Up to 2,000 m (6,562 ft)
Pollution degree 2
Humidity 20% to 85% relative non-condensing
Air filtering No excessive dust or smoke
Lighting No special requirements

Chapter 12: Specifications 141

Loader

Item Specification

Dimensions 23 x 47 x 28 cm (9.1 x 18.5 x 11 in)

(W x D x H)
Weight AMS: 13 kg (28.7 lb)
ASL: 15 kg (33.1 lb)
Power 160 W (24 V, 6.67 A)
Connection USB A to USB A
Throughput Default
modes Low Carryover
High Throughput
Throughput time AMS: 35 min for High Throughput mode, sampling 7 µL/well from 96-well plate
ASL: 27 min for High Throughput mode, sampling 7 µL/well from 96-well plate
Dead volume <10 µL for 96-well flat-bottom plates
<5 µL for all other supported plate types
Carryover Default mode: 0.3%
Low Carryover mode: 0.1%
High Throughput mode: 1%

Supported Carrier Types

The system supports the following carrier types:

Carrier Material Part Number

96-well flat-bottom Polypropylene VWR 29444-100

Polystyrene VWR 29442-070
96-well U-bottom Polypropylene VWR 29444-104
Polystyrene VWR 29442-396
96-well V-bottom Polypropylene VWR 29444-102
Polystyrene VWR 29442-402
Nunc 1.3 mL U96 DeepWella Polypropylene VWR 736-0601
Nunc 2.0 mL U96 DeepWella Polypropylene VWR 736-0607
BRAND 1 mL 96-well deep wella Polypropylene VWR 80087-068
Polystyrene
BRAND 2.2 mL 96-well deep wella Polypropylene
Cytek 40-tube racka N/A N/A
a. Supported on the ASL only.

142 Aurora User’s Guide

 13
Supplies and Replacement Parts

Item Part Number Description

4-L Tank N4-00124 4-L tank for sheath or waste

4-L supply cap assembly N7-32015 Lid fits 4-L sheath tank
4-L waste cap assembly N7-32014 Lid fits 4-L waste tank and includes liquid level
sensor
20-L cubitainer adapter N7-32016 Includes fluidics lines and waste liquid level sensor
assembly
Reservoir holder N4-00128 Holds the sheath and waste tanks
SpectroFlo QC Beads B7-10001 2 mL of beads at 107/mL concentration. Beads
2000 series, 2 mL (formerly provide a single peak fluorescence intensity for use
N7-97355) with Daily QC.
Sheath filter assembly N7-22006 0.2-µm sheath filter assembly with quick-connect
(0.2 µm) fittings and manifold
Sheath filter assembly N7-22016 0.4-µm sheath filter assembly with quick-connect
(0.4 µm) fittings and manifold
Sample line N7-22014 SIT tubing assembly
Sheath filter bypass N7-22010 Replaces the sheath filter during a Long Clean
(Long Clean tubing)
25-mm disk filter N4-00047 Filter at sheath tubing intake for cubitainer and 4-L
(20 µm) sheath tank cap

Chapter 13: Supplies and Replacement Parts 143

144 Aurora User’s Guide
 Index

A raw 43, 68
accumulator vessel 14, 15 unmixed 43, 68, 73
acquisition degasser 14
controls 45 deleting
controls, loader 112 user account 103
experiments 44
preferences 88 E
adding editing
fluorescent tags to library 80 fluorescent tag properties 80
labels 81 gate properties 50
new user account 102 plot properties 49
alarm ranges 41 user account 102
Analyzing Data Offline 68 experiment
about 18
area scaling factor 47
creating new 53–60
B default 18, 21
display 44–51
benchmark reference controls 39
exporting 19
C in plate mode 114
setup overview 44
cleaning
templates 19, 85
external surfaces 126
toolbar 48
flow cell 123
SIT 122 export
experiment 19
complexity index 67
QC report 32
cytometer
cleaning surfaces 126 F
fluid containers 14, 25–26 FCS files
fluidics system components 14–15 formats 19, 43
lasers 16 raw data 68
optics 15 storage preferences 97
overview 12 unmixed data 68, 73
power button 12
flow cell, cleaning 123
replacement parts 143
sheath filter 15 flow meter 128
shutdown 28 fluid containers 14, 25–26
SIT/SIP 13 fluidics lines
startup 27 inspecting 126
fluidics system
D components 14–15
daily QC decontaminating 123
performing 29–32 specifications 140
report 32 fluorescent tag
data adding new 80
formats 43 editing properties 80

145
 groups 34 long clean 123
library 79
live unmixing 65 M
running reference controls 34 maintenance
selecting in experiment 54 scheduled 121
font preferences 95 unscheduled 121
modules, software 17
G
gain N
adjusting 47 notification preferences 96
See also user settings
settings 41 O
gates optics 15
adjusting, post-acquisition unmixing 69, 72 compartment 12
boolean 50 specifications 139
preferences 93 overview
properties 50 Aurora system 11
cytometer 12
I loader 109
installation requirements 141 software 17
unmixing 62
K
keywords 81 P
password, resetting 103
L plate, 96-well
labels calibrating 118
adding 81 loading 110
library 80 stage temperature 109
lasers supported types 142
detectors 16 plenum 14
scaling factor 47 plots
library preferences 91
experiment templates 85 types and properties 48
fluorescent tags 79 post-acquisition unmixing
keywords 81 in acquisition module 69–??
labels 80 in analysis module 70, 70–73
loader settings 84
power button 12
QC beads 79
user settings 83 preferences
worksheet templates 84 acquisition 88
fonts 95
loader
gates 93
calibrating plate 118
notifications 96
calibrating SIT 119
plot 91
enabling 110
QC setup 98
loading a plate 110
statistics 94
mixing probe 109
storage 97
overview 109
worksheet 90
plate stage temperature 109
setting sample delivery method 99 Q
settings 84, 116
QC beads
specifications 142
library 79
wash station 109

146 Aurora User’s Guide

 running 29–32 overview 17
QC reports spillover
exporting 32 adjusting 51
pass/fail criteria 32 defined 61
QC setup starting up system 27
preferences 98 statistics
creating 50
R preferences 94
reference controls 34 system overview 11
comparing to benchmarks 66
running 34–38 T
selecting benchmarks 39 technical support information 10
updating 39
threshold 47
replacement parts 143
toolbar, experiment 48
replacing
troubleshooting 131–133
sheath filter 126
SIT 127–130 U
S unmixing 61
label/lot specific 89
safety
live 64–68
biological 9
overview 62
electrical 8
post-acquisition 69–73
general information 7
symbols 7 user settings 41
library 83
sample injection port (SIP) 13
users
sample injection tube (SIT) 13
adding new account 102
calibrating for loader 119
editing account 102
compartment 12
removing account 103
flushing 122
resetting user password 103
flushing, loader 114
use time on system 103
replacing 127–130
settings V
loader 113, 116 virtual filters 74–78
user 41
sheath container 14 W
filling 25–26 waste container 14
sheath filter 15 emptying 26
purging 122 worksheet
replacing 126 about 21
sheath plenum 14 preferences 90
shutting down system 28 selecting 48
signal measurements 47 templates 84
similarity index 66 workstation specifications 141
SIP
See sample injection port
SIT
See sample injection tube
software
FCS file formats 43
modules 17

147
148 Aurora User’s Guide
Cytek Biosciences, Inc
46107 Landing Pkwy.
Fremont, CA 94538
1.877.92.CYTEK (1.877.922.9835)

[email protected]
cytekbio.com

N9-20006 Rev. D
July 2021