BD Cytometric Bead Array

(CBA) Mouse Th1/Th2/Th17

Cytokine Kit
Instruction Manual

Catalog No. 560485

ii BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Trademarks
FCAP Array is a registered trademark of Softflow, Inc.

Macintosh and Mac are trademarks of Apple Computer, Inc., registered in the US and other
countries.

Falcon® is a registered trademark of Corning Incorporated.

History

Revision Date Change made

647209 2/2009 Initial document

23-12380-00 Rev. 01 10/2010 Setup and acquisition instructions removed

Rev. 2 4/2015 Update hazard statements

BD flow cytometers are Class 1 Laser Products.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
© 2014 Becton, Dickinson and Company. All rights reserved. No part of this publication may be
reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or
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Purchase does not include or carry any right to resell or transfer this product either as a stand-alone
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BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2014 BD
Contents

Chapter 1: About this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Purpose of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Storage and handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Chapter 2: Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapter 3: Assay preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Preparing Mouse Th1/Th2/Th17 Cytokine Standards . . . . . . . . . . . 18
Mixing Mouse Th1/Th2/Th17 Cytokine Capture Beads . . . . . . . . . 20
Diluting samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Chapter 4: Assay procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Performing the Mouse Th1/Th2/Th17 Cytokine Assay . . . . . . . . . . 24
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Chapter 5: Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Theoretical limit of detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Linearity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Chapter 6: Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 1
About this kit
This section covers the following topics:

 Purpose of the kit (page 6)

 Limitations (page 8)
 Kit contents (page 9)
 Storage and handling (page 11)

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
6 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Purpose of the kit

Use of the kit The BD™ CBA Mouse Th1/Th2/Th17 Cytokine Kit can
be used to measure Interleukin-2 (IL-2), Interleukin-4
(IL-4), Interleukin-6 (IL-6), Interferon- (IFN-), Tumor
Necrosis Factor (TNF), Interleukin-17A (IL-17A), and
Interleukin-10 (IL-10) protein levels in a single sample.
The kit performance has been optimized for analysis of
physiologically relevant concentrations (pg/mL levels) of
specific cytokine proteins in tissue culture supernatants
and serum samples. The kit provides sufficient reagents
for 80 tests.

Principle of CBA BD CBA assays provide a method of capturing a soluble

assays analyte or set of analytes with beads of known size and
fluorescence, making it possible to detect analytes using
flow cytometry.

Each capture bead in a BD CBA kit has been conjugated

with a specific antibody. The detection reagent provided
in the kit is a mixture of phycoerythrin (PE)–conjugated
antibodies, which provides a fluorescent signal in
proportion to the amount of bound analyte.

When the capture beads and detector reagent are

incubated with an unknown sample containing
recognized analytes, sandwich complexes (capture bead
+ analyte + detection reagent) are formed. These
complexes can be measured using flow cytometry to
identify particles with fluorescence characteristics of
both the bead and the detector.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 1: About this kit 7

Principle of this The BD CBA BD CBA Mouse Th1/Th2/Th17 Cytokine

assay Kit uses bead array technology to simultaneously detect
multiple cytokine proteins in research samples. Seven
bead populations with distinct fluorescence intensities
have been coated with capture antibodies specific for IL-
2, IL-4, IL-6, IFN-, TNF, IL-17A, and IL-10 proteins.
The seven bead populations are mixed together to form
the bead array, which is resolved in a red channel (ie,
FL3 or FL4) of a flow cytometer.

Figure 1

During the assay procedure, you will mix the cytokine

capture beads with recombinant standards or unknown
samples and incubate them with the PE-conjugated
detection antibodies to form sandwich complexes. The
intensity of PE fluorescence of each sandwich complex
reveals the concentration of that cytokine. After
acquiring samples on a flow cytometer, use FCAP
Array™ software to generate results in graphical and
tabular format.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
8 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Advantages over The broad dynamic range of fluorescent detection via

ELISA flow cytometry and the efficient capturing of analytes via
suspended particles enable the BD CBA assay to measure
the concentration of an unknown in substantially less
time and using fewer sample dilutions compared to
conventional ELISA methodology.

 The required sample volume is approximately one-

seventh the quantity necessary for conventional
ELISA assays due to the detection of seven analytes
in a single sample.
 A single set of diluted standards is used to generate a
standard curve for each analyte.
 A BD CBA experiment takes less time than a single
ELISA and provides results that would normally
require seven conventional ELISAs.

Limitations
Assay limitations The theoretical limit of detection of the BD CBA BD
CBA Mouse Th1/Th2/Th17 Cytokine Kit is comparable
to conventional ELISA, but due to the complexity and
kinetics of this multi-analyte assay, the actual limit of
detection on a given experiment may vary. See
Theoretical limit of detection (page 30) and Precision
(page 35).

The BD CBA Kit is not recommended for use on stream-

in-air instruments for which signal intensities may be
reduced, adversely affecting assay sensitivity. Stream-in-
air instruments include the BD FACStar™ Plus,
BD FACSVantage™, and BD Influx™ flow cytometers
(BD Biosciences).

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 1: About this kit 9

Serum spike recoveries for IL-4 and TNF are lower than
for the other cytokines in this assay. This variation is due
to assay conditions and serum proteins and may affect
quantitation of these proteins in serum samples.

Quantitative results or protein levels for the same sample

or recombinant protein run in ELISA and BD CBA
assays may differ. A spike recovery assay can be
performed using an ELISA standard followed by BD
CBA analysis to assess possible differences in
quantitation.

This kit is designed to be used as an integral unit. Do not

mix components from different batches or kits.

Kit contents
Contents The BD CBA Mouse Th1/Th2/Th17 Cytokine Kit
contains the following components sufficient for 80
tests.

Vial
label Reagent Quantity
A1 Mouse IL-2 Capture Beads 1 vial, 0.8 mL
A2 Mouse IL-4 Capture Beads 1 vial, 0.8 mL
A3 Mouse IL-6 Capture Beads 1 vial, 0.8 mL
A4 Mouse IFN- Capture Beads 1 vial, 0.8 mL
A5 Mouse TNF Capture Beads 1 vial, 0.8 mL
A6 Mouse IL-17A Capture Beads 1 vial, 0.8 mL
A7 Mouse IL-10 Capture Beads 1 vial, 0.8 mL
B Mouse Th1/Th2/Th17 PE 1 vial, 4 mL
Detection Reagent

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
10 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Vial
label Reagent Quantity
C Mouse Th1/Th2/Th17 2 vials
Cytokine Standards lyophilized
D Cytometer Setup Beads 1 vial, 1.5 mL
E1 PE Positive Control Detector 1 vial, 0.5 mL
E2 FITC Positive Control Detector 1 vial, 0.5 mL
F Wash Buffer 1 bottle, 130 mL
G Assay Diluent 1 bottle, 30 mL

Bead reagents Mouse Cytokine Capture Beads (A1–A7): An 80-test

vial of each specific capture bead (A1–A7). The specific
capture beads, having discrete fluorescence intensity
characteristics, are distributed from brightest (A1) to
dimmest (A7).

Cytometer Setup Beads (D): A 30-test vial of setup beads

for setting the initial instrument PMT voltages and
compensation settings is sufficient for 10 instrument
setup procedures. The Cytometer Setup Beads are
formulated for use at 50 µL/test.

Antibody and Mouse Th1/Th2/Th17 PE Detection Reagent (B): An

standard 80-test vial of PE-conjugated anti-mouse IL-2, IL-4, IL-6,
reagents IFN, TNF, IL-17A, and IL-10 antibodies that is
formulated for use at 50 µL/test.

Mouse Th1/Th2/Th17 Cytokine Standards (C): Two

vials containing lyophilized recombinant mouse cytokine
proteins. Each vial should be reconstituted in 2.0 mL of
Assay Diluent to prepare the top standard.

PE Positive Control Detector (E1): A 10-test vial of PE-

conjugated antibody control that is formulated for use at
50 µL/test. This reagent is used with the Cytometer Setup
Beads to set the initial instrument compensation settings.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 1: About this kit 11

FITC Positive Control Detector (E2): A 10-test vial of

FITC-conjugated antibody control that is formulated for
use at 50 µL/test. This reagent is used with the
Cytometer Setup Beads to set the initial instrument
compensation settings.

Buffer reagents Wash Buffer (F): A 130-mL bottle of phosphate buffered

saline (PBS) solution (1X), containing protein and
detergent, used for wash steps and to resuspend the
washed beads for analysis.

Assay Diluent (G): A 30-mL bottle of a buffered protein

solution (1X) used to reconstitute and dilute the Mouse
Th1/Th2/Th17 Cytokine Standards and to dilute
unknown samples.

Note: Source of all serum proteins is from USDA

inspected abattoirs located in the United States.

Storage and handling

Storage Store all kit components at 2 to 8°C. Do not freeze.

Warning Components A1–A7, B, D, E1, E2, F, and G contain

sodium azide. Sodium azide yields highly toxic hydrazoic
acid under acidic conditions. Dilute azide compounds in
running water before discarding to avoid accumulation
of potentially explosive deposits in plumbing.

Mouse Th1/Th2/Th17 Cytokine Standards (component

51-9006250) contains 0.02% (w/w) of a CMIT/MIT
mixture (3:1), which is a mixture of: 5-chloro-2-methyl-
4-isothiazolin-3-one [EC No 247-500-7] and 2-methyl-
4-isothiazolin-3-one [EC No 220-239-6] (3:1).

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
12 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Hazard May cause an allergic skin reaction.

statements

Precautionary Wear protective gloves / eye protection.

statements
Wear protective clothing.

Avoid breathing mist/vapours/spray.

If skin irritation or rash occurs: Get medical advice/

attention.

IF ON SKIN: Wash with plenty of water.

Dispose of contents/container in accordance with local/

regional/national/international regulations.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 2
Before you begin
This section covers the following topics:

 Workflow overview (page 14)

 Required materials (page 15)

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
14 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Workflow overview
Workflow The overall workflow consists of the following steps:

Step Description
1 Preparing Mouse Th1/Th2/Th17 Cytokine
Standards (page 18)
2 Mixing Mouse Th1/Th2/Th17 Cytokine Capture
Beads (page 20)
3 Diluting samples (page 21), if necessary
4 Performing instrument setup with Cytometer
Setup Beads (instructions can be found at
bdbiosciences.com/cbasetup)
Note: Can be performed during the incubation in
step 5.
5 Performing the Mouse Th1/Th2/Th17 Cytokine
Assay (page 24)
6 Acquiring samples (instructions can be found at
bdbiosciences.com/cbasetup)
7 Data analysis (page 27)

Incubation times To help you plan your work, the incubation times are
listed in the following table:

Procedure Incubation time

Preparing standards 15 minutes
Preparing Cytometer Setup Beads 30 minutes
Performing the assay 2 hours

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 2: Before you begin 15

Required materials
Materials In addition to the reagents provided in the BD CBA BD
required but not CBA Mouse Th1/Th2/Th17 Cytokine Kit, the following
provided items are also required:

 A dual-laser flow cytometer equipped with a 488-

nm or 532-nm and a 633-nm or 635-nm laser
capable of distinguishing 576-nm, 660-nm, and
>680-nm fluorescence. The following table lists
examples of compatible instrument platforms.

Reporter
Flow cytometer channel Bead channels
BD FACSArray™ Yellow Red
BD FACSCanto™ platform
BD™ LSR platform
BD FACSAria™ platform PE APC
BD FACSCalibur™ (single laser) FL3
BD FACSCalibur (dual laser) FL2 FL4
Note: Visit bdbiosciences.com/cbasetup for setup protocols.

 Falcon® 12 × 75-mm sample acquisition tubes

(Catalog No. 352008), or equivalent
 15-mL conical, polypropylene tubes (Falcon,
Catalog No. 352097), or equivalent
 FCAP Array software (Catalog No. 641488 [PC] or
645447 [Mac])

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
16 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Materials  Millipore MultiScreenHTS-BV 1.2 µm Clear non-

required for sterile filter plates [Catalog No. MSBVN1210 (10
plate loader- pack) or MSBVN1250 (50 pack)]
equipped flow
 Millipore MultiScreenHTS Vacuum Manifold,
cytometers
(Catalog No. MSVMHTS00)
 MTS 2/4 Digital Stirrer, IKA Works, VWR (Catalog
No. 82006-096)
 Vacuum source
 Vacuum gauge and regulator (if not using the
recommended manifold)

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 3
Assay preparation
This section covers the following topics:

 Preparing Mouse Th1/Th2/Th17 Cytokine Standards (page 18)

 Mixing Mouse Th1/Th2/Th17 Cytokine Capture Beads (page 20)
 Diluting samples (page 21)

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
18 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Preparing Mouse Th1/Th2/Th17 Cytokine Standards

Purpose of this The Mouse Th1/Th2/Th17 Cytokine Standards are
procedure lyophilized and should be reconstituted and serially
diluted immediately before mixing with the Capture
Beads and the PE Detection Reagent.

You must prepare fresh cytokine standards to run with

each experiment. Do not store or reuse reconstituted or
diluted standards.

Procedure To reconstitute and serially dilute the standards:

1. Open one vial of lyophilized Mouse Th1/Th2/Th17
Standards. Transfer the standard spheres to a 15-mL
conical, polypropylene tube. Label the tube “Top
Standard.”
2. Reconstitute the standards with 2.0 mL of Assay
Diluent.
a. Allow the reconstituted standard to equilibrate
for at least 15 minutes at room temperature.
b. Gently mix the reconstituted protein by pipette
only. Do not vortex or mix vigorously.
3. Label 12 × 75-mm tubes and arrange them in the
following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64,
1:128, and 1:256.
4. Pipette 300 µL of Assay Diluent in each of the 12 ×
75-mm tubes.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 3: Assay preparation 19

5. Perform serial dilutions:

a. Transfer 300 µL from the Top Standard to the
1:2 dilution tube and mix thoroughly by pipette
only.
b. Continue making serial dilutions by transferring
300 µL from the 1:2 tube to the 1:4 tube and so
on to the 1:256 tube.
c. Mix thoroughly by pipette only. Do not vortex.

6. Prepare one 12 × 75-mm tube containing only Assay

Diluent to serve as the 0 pg/mL negative control.

Concentration of See the Procedure for tubes section of Performing the

standards Mouse Th1/Th2/Th17 Cytokine Assay (page 24) for a
listing of the concentrations (pg/mL) of all seven
recombinant proteins in each standard dilution.

Next step Proceed to Mixing Mouse Th1/Th2/Th17 Cytokine

Capture Beads (page 20).

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
20 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Mixing Mouse Th1/Th2/Th17 Cytokine Capture Beads

Purpose of this The Capture Beads are bottled individually (A1–A7).
procedure You must pool all seven bead reagents immediately
before using them in the assay.

Procedure To mix the Capture Beads:

1. Determine the number of assay tubes (including
standards and controls) required for the experiment
(eg, 8 unknowns, 9 cytokine standard dilutions, and
1 negative control = 18 assay tubes).
2. Vigorously vortex each Capture Bead suspension for
3 to 5 seconds before mixing.
Note: The antibody-conjugated beads will settle out
of suspension over time. It is necessary to vortex the
vial before taking a bead-suspension aliquot.

3. Add a 10-µL aliquot of each Capture Bead, for each

assay tube to be analyzed, into a single tube labeled
“mixed Capture Beads” (eg, 10 µL of IL-2 Capture
Beads × 18 assay tubes = 180 µL of IL-2 Capture
Beads required).
4. Vortex the bead mixture thoroughly.

Next step The mixed Capture Beads are now ready to be

transferred to the assay tubes. Discard excess mixed
Capture Beads. Do not store after mixing.

To begin the assay, proceed to Performing the Mouse

Th1/Th2/Th17 Cytokine Assay (page 24). If you need to
dilute samples having a high cytokine concentration,
proceed to Diluting samples (page 21).

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 3: Assay preparation 21

Diluting samples
Purpose of this The standard curve for each cytokine covers a defined set
procedure of concentrations from 20 to 5000 pg/mL. It might be
necessary to dilute samples to ensure that their mean
fluorescence values fall within the range of the generated
cytokine standard curve. For best results, dilute samples
that are known or assumed to contain high levels of a
given cytokine. This procedure is not necessary for all
samples.

Procedure To dilute samples with a known high cytokine

concentration:
1. Dilute the sample by the desired dilution factor (ie,
1:2, 1:10, or 1:100) using the appropriate volume of
Assay Diluent.
2. Mix sample dilutions thoroughly.

Next step Perform instrument setup using the Cytometer Setup

Beads. For details, go to bdbiosciences.com/cbasetup and
select the appropriate flow cytometer under CBA Kits:
Instrument Setup.

Or, if you wish to begin staining your samples for the

assay, proceed to Performing the Mouse Th1/Th2/Th17
Cytokine Assay (page 24), and you can perform
instrument setup during the 2-hour staining incubation.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 4
Assay procedure
This section covers the following topics:

 Performing the Mouse Th1/Th2/Th17 Cytokine Assay (page 24)

 Data analysis (page 27)

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
24 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Performing the Mouse Th1/Th2/Th17 Cytokine Assay

Before you begin  Prepare the standards as described in Preparing
Mouse Th1/Th2/Th17 Cytokine Standards
(page 18).
 Mix the Capture Beads as described in Mixing
Mouse Th1/Th2/Th17 Cytokine Capture Beads
(page 20).
 If necessary, dilute the unknown samples. See
Diluting samples (page 21).

Procedure for To perform the assay:

tubes 1. Vortex the mixed Capture Beads and add 50 µL to
all assay tubes.
2. Add 50 µL of the Mouse Th1/Th2/Th17 Cytokine
Standard dilutions to the control tubes as listed in
the following table.

Concentration Cytokine Standard

Tube label (pg/mL) dilution
1 0 (negative control) no standard dilution
(Assay Diluent only)
2 20 1:256
3 40 1:128
4 80 1:64
5 156 1:32
6 312.5 1:16
7 625 1:8
8 1,250 1:4
9 2,500 1:2
10 5,000 Top standard

3. Add 50 µL of each unknown sample to the

appropriately labeled sample assay tubes.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 4: Assay procedure 25

4. Add 50 µL of the Mouse Th1/Th2/Th17 PE

Detection Reagent to all assay tubes.
5. Incubate the assay tubes for 2 hours at room
temperature, protected from light.
Note: If you have not yet performed cytometer
setup, you may wish to do so during this incubation.

6. Add 1 mL of Wash Buffer to each assay tube and

centrifuge at 200g for 5 minutes.
7. Carefully aspirate and discard the supernatant from
each assay tube.
8. Add 300 µL of Wash Buffer to each assay tube to
resuspend the bead pellet.

Procedure for To perform the assay:

filter plates 1. Wet the plate by adding 100 µL of wash buffer to
each well.
2. Place the plate on the vacuum manifold.
3. Aspirate for 2 to 10 seconds until the wells are
drained.
4. Remove the plate from the manifold, then blot the
bottom of the plate on paper towels.
5. Add 50 µL of each of the following to the wells in
the filter plate:
• Capture Beads (vortex before adding)
• Standard or sample (add standards from the
lowest concentration to the highest, followed by
samples)
• Mouse Th1/Th2/Th17 PE Detection Reagent
6. Cover the plate and shake it for 5 minutes at 1,100
rpm on a plate shaker.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
26 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

7. Incubate the plate for 2 hours at room temperature

on a non-absorbent, dry surface.
Note: Place the plate on a non-absorbent, dry
surface during incubation. Absorbent or wet
surfaces can cause the contents of the wells to leak.

8. Remove the cover from the plate and apply the plate
to the vacuum manifold.
9. Vacuum aspirate for 2 to 10 seconds until the wells
are drained.
10. Remove the plate from the manifold, then blot the
bottom of the plate on paper towels after aspiration.
11. Add 120 µL of wash buffer to each well to resuspend
the beads.
12. Cover the plate and shake it for 2 minutes at 1,100
rpm before you begin sample acquisition.

Next step Acquire the samples on the flow cytometer. For details,
go to bdbiosciences.com/cbasetup and select the
appropriate flow cytometer under CBA Kits: Instrument
Setup.

Acquire samples on the same day they are prepared.

Prolonged storage of samples, once the assay is
complete, can lead to increased background and reduced
sensitivity.

To facilitate the analysis of samples using FCAP Array

software, we recommend the following guidelines:

 Acquire standards from lowest (0 pg/mL) to highest

(Top Standard) concentration, followed by the test
samples.
 If running sample dilutions, acquire sequentially
starting with the most concentrated sample.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 4: Assay procedure 27

 Store all FCS files (standards and samples) in a single

folder.
When you are finished acquiring samples, proceed to
Data analysis (page 27).

Data analysis
How to analyze Analyze Mouse Th1/Th2/Th17 Cytokine data using
FCAP Array software. For instructions on analysis, go to
bdbiosciences.com/cbasetup and see the Guide to
Analyzing Data from BD CBA Kits Using FCAP Array
Software.

Typical data The following data, acquired using BD CellQuest

software, shows standards and detectors alone.

Negative control (0 pg/mL) Standard 80 pg/mL

Standard 625 pg/mL Standard 5000 pg/mL

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
28 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Standard curve The following graphs represent standard curves from the
examples Mouse Th1/Th2/Th17 Cytokine Standards.

IL-2 IL-4 IL-6

IFN- TNF IL-17A

IL-10

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 5
Performance
This section covers the following topics:

 Theoretical limit of detection (page 30)

 Recovery (page 31)
 Linearity (page 32)
 Specificity (page 33)
 Precision (page 35)

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
30 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Theoretical limit of detection

Experiment The individual standard curve range for a given cytokine
details defines the minimum and maximum quantifiable levels
using the BD CBA Mouse Th1/Th2/Th17 Cytokine Kit
(ie, 20 pg/mL and 5000 pg/mL). By applying the 4-
parameter curve fit option, it is possible to extrapolate
values for sample intensities not falling within the limits
of the standard curve. It is up to the researcher to decide
the best method for calculating values for unknown
samples using this assay. The theoretical limit of
detection for each cytokine using the BD CBA Mouse
Th1/Th2/Th17 Cytokine Kit is defined as the
corresponding concentration at two standard deviations
above the median fluorescence of 30 replicates of the
negative control (0 pg/mL).

Limit of
detection data Cytokine Limit of detection (pg/mL)
IL-2 0.1
IL-4 0.03
IL-6 1.4
IFN- 0.5
TNF 0.9
IL-17A 0.8
IL-10 16.8

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 5: Performance 31

Recovery
Experiment Individual cytokine protein was spiked into various
details matrices at three different levels within the assay range.
The cell culture medium used in these experiments was
not diluted before addition of the cytokine protein. The
pooled mouse serum samples in these experiments were
diluted 1:4 in Assay Diluent before addition of the
cytokine protein. The spiked samples were assayed and
the results were compared with the expected values.

Recovery data
Average %
Cytokine Matrix Recovery Range (%)
IL-2 Media 83 81–87
Serum 81 79–85
IL-4 Media 83 79–91
Serum 38 35–45
IL-6 Media 83 83–84
Serum 79 77–83
IFN- Media 84 81–86
Serum 76 72–82
TNF Media 97 94–102
Serum 69 63–76
IL-17A Media 82 80–85
Serum 61 58–63
IL-10 Media 97 78–121
Serum 78 74–80

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
32 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Linearity
Experiment In two experiments, the following matrices were spiked
details with IL-2, IL-4, IL-6, IFN-, TNF, IL-17A, and IL-10 and
then were serially diluted with Assay Diluent.

Linearity data
Sample Detected Average %
Cytokine Matrix dilution (pg/mL) of expected
IL-2 Media 1:2 1089.6 100
1:4 490.3 90
1:8 244.3 90
Serum 1:2 1045.1 100
1:4 491.9 94
1:8 250.0 96
IL-4 Media 1:2 1124.2 100
1:4 496.5 88
1:8 251.1 89
Serum 1:2 549.2 100
1:4 324.8 118
1:8 189.3 138
IL-6 Media 1:2 1008.5 100
1:4 496.1 98
1:8 245.4 97
Serum 1:2 957.8 100
1:4 476.7 100
1:8 242.0 101
IFN- Media 1:2 1017.5 100
1:4 503.7 99
1:8 263.3 104
Serum 1:2 891.2 100
1:4 480.2 108
1:8 256.0 115

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 5: Performance 33

Sample Detected Average %

Cytokine Matrix dilution (pg/mL) of expected
TNF Media 1:2 1208.3 100
1:4 572.6 95
1:8 295.2 98
Serum 1:2 777.9 100
1:4 417.3 107
1:8 222.4 114
IL-17A Media 1:2 999.5 100
1:4 450.4 90
1:8 234.0 94
Serum 1:2 735.1 100
1:4 418.0 114
1:8 230.6 125
IL-10 Media 1:2 959.5 100
1:4 490.5 102
1:8 294.0 123
Serum 1:2 833.3 100
1:4 485.2 116
1:8 270.9 130

Specificity
Experiment The antibodies used in the BD CBA Mouse Th1/Th2/
details Th17 Cytokine Kit have been screened for specific
reactivity with their specific cytokines. Analysis of
samples containing only a single recombinant cytokine
protein found no cross-reactivity or background
detection of cytokine in other Capture Bead populations
using this assay.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
34 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Specificity data Sample data containing only a single recombinant

cytokine protein was acquired using BD CellQuest
software.

Mouse IL-2 Mouse IL-4 Mouse IL-6

Mouse IFN- Mouse TNF Mouse IL-17A

Mouse IL-10

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 5: Performance 35

Precision
Intra-assay Ten replicates of each of three different levels of IL-2, IL-
precision 4, IL-6, IFN-, TNF, IL-17A, and IL-10 were tested.

Mean Standard
Cytokine Sample (pg/mL) deviation %CV
Sample 1 71.1 1.7 2
IL-2 Sample 2 292.0 7.9 3
Sample 3 1238.7 42.4 3
Sample 1 70.6 2.6 4
IL-4 Sample 2 291.8 7.8 3
Sample 3 1357.9 39.7 3
Sample 1 79.1 4.3 5
IL-6 Sample 2 315.0 18.1 6
Sample 3 1206.8 35.6 3
Sample 1 79.6 1.9 2
IFN- Sample 2 313.7 9.6 3
Sample 3 1233.6 39.9 3
Sample 1 77.9 3.7 5
TNF Sample 2 291.6 10.7 4
Sample 3 1172.2 45.4 4
Sample 1 82.8 2.8 3
IL-17A Sample 2 316.6 7.8 2
Sample 3 1253.2 28.8 2
Sample 1 89.5 18.7 21
IL-10 Sample 2 307.5 34.8 11
Sample 3 1115.3 39.1 4

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
36 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Inter-assay Three different levels of IL-2, IL-4, IL-6, IFN-, TNF, IL-
precision 17A, and IL-10 were tested in four experiments
conducted by four different operators.

Mean Standard
Cytokine Sample (pg/mL) deviation %CV
IL-2 Sample 1 76.0 3.5 5
Sample 2 299.2 10.0 3
Sample 3 1283.2 44.1 3
IL-4 Sample 1 76.2 4.5 6
Sample 2 299.1 8.5 3
Sample 3 1333.1 85.0 6
IL-6 Sample 1 81.4 4.6 6
Sample 2 314.7 13.8 4
Sample 3 1228.4 47.7 4
IFN- Sample 1 81.1 2.9 4
Sample 2 313.7 9.8 3
Sample 3 1240.8 39.2 3
TNF Sample 1 82.7 5.8 7
Sample 2 298.8 13.3 4
Sample 3 1224.5 65.4 5
IL-17A Sample 1 84.4 3.6 4
Sample 2 316.1 7.5 2
Sample 3 1252.0 54.9 4
IL-10 Sample 1 94.8 19.4 20
Sample 2 342.6 37.9 11
Sample 3 1197.7 66.9 6

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 6
Reference
This section covers the following topics:

 Troubleshooting (page 38)

 References (page 39)

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
38 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Troubleshooting
Recommended These are the actions we recommend you take if you
actions encounter the following problems.

Problem Recommended actions

Variation between Vortex Capture Beads before pipetting. Beads can
duplicate samples aggregate.
Low bead number in Avoid aspiration of beads during wash step. Do not
samples wash or resuspend beads in volumes higher than
recommended volumes.
High background Test various sample dilutions. The sample might be too
concentrated. Remove excess Mouse Th1/Th2/TH17
PE Detection Reagent by increasing the number of
wash steps, since background may be due to non-
specific binding.
Little or no protein Sample may be too dilute. Try various sample dilutions.
detected in sample
Less than seven bead Ensure that equal volumes of beads were added to each
populations observed assay tube. Vortex Capture Bead vials before taking
during analysis, or aliquots. Once Capture Beads are mixed, vortex to
distribution is unequal ensure beads are distributed throughout solution.
Debris (FSC/SSC) Increase FSC threshold or further dilute samples.
during sample Increase number of wash steps, if necessary.
acquisition
Overlap of bead Samples might have very high cytokine concentration.
fluorescence (FL3) Ensure instrument settings have been optimized using
during acquisition Cytometer Setup Beads.
Standards show low Ensure that all components are properly prepared and
fluorescence or poor stored. Use new vial of standard with each experiment
standard curve and once reconstituted, do not use after 12 hours.
Ensure that incubation times were of proper length.
All samples are positive Dilute samples further. Samples might be too
or above high standard concentrated.
mean fluorescence value

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 6: Reference 39

References
Related 1. Bishop JE, Davis KA. A flow cytometric
publications immunoassay for 2-microglobulin in whole blood.
J Immunol Methods. 1997;210:79–87.
2. Camilla C, Defoort JP, Delaage M, Auer R,
Quintana J, Lary T, Hamelik R, Prato S, Casano B,
Martin M, Fert V. A new flow cytometry-based
multi-assay system. 1. Application to cytokine
immunoassays. Cytometry Suppl. 1998;8:132.
3. Carson R, Vignali D. Simultaneous quantitation of
fifteen cytokines using a multiplexed flow cytometric
assay. J Immunol Methods. 1999;227:41–52.
4. Chen R, Lowe L, Wilson JD, et al. Simultaneous
quantification of six human cytokines in a single
sample using microparticle-based flow cytometric
technology. Clin Chem. 1999;45:1693–1694.
5. Collins DP, Luebering BJ, Shaut DM. T-lymphocyte
functionality assessed by analysis of cytokine
receptor expression, intracellular cytokine
expression, and femtomolar detection of cytokine
secretion by quantitative flow cytometry. Cytometry.
1998;33:249–255.
6. Fulton RJ, McDade RL, Smith PL, Kienker LJ,
Kettman JR Jr. Advanced multiplexed analysis with
the FlowMetrix system. Clin Chem. 1997;43:1749–
1756.
7. Kricka LJ. Simultaneous multianalyte
immunoassays. In Immunoassay. Diamandis EP,
Christopoulos TK, eds. Academic Press. 1996:389–
404.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
40 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

8. Lund-Johansen F, Davis K, Bishop J, de WaalMalefyt

R. Flow cytometric analysis of immunoprecipitates:
High-throughput analysis of protein
phosphorylation and protein-protein interactions.
Cytometry. 2000;39:250–259.
9. McHugh TM. Flow microsphere immunoassay for
the quantitative and simultaneous detection of
multiple soluble analytes. Methods Cell Biol.
1994;42:575–595.
10. Oliver KG, Kettman JR, Fulton RJ. Multiplexed
analysis of human cytokines by use of the
FlowMetrix system. Clin Chem. 1998;44:2057–
2060.
11. Stall A, Sun Q, Varro R, Lowe L, Crowther E,
Abrams B, Bishop J, Davis K. A single tube flow
cytometric multibead assay for isotyping mouse
monoclonal antibodies. Abstract LB77.
Experimental Biology Meeting 1998 (late-breaking
abstracts).

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
 Chapter 6: Reference 41

Notes

For Research Use Only. Not for use in diagnostic or therapeutic procedures.
42 BD CBA Mouse Th1/Th2/Th17 Cytokine Kit

Notes

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