ab65475
Quick Cell Proliferation
Assay Kit II
Instructions for Use
For the rapid, sensitive and accurate
measurement of Cell Proliferation in cell culture
(adherent and suspension)
This product is for research use only and is not
intended for diagnostic use.
Version: 2 Last Updated: 23/08/2013
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Table of Contents
1. Overview 3
2. Protocol Summary 4
3. Components and Storage 5
4. Assay Protocol 7
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1. Overview
Abcam’s Quick Cell Proliferation Assay Kit II provides by far the
easiest and most sensitive means for quantifying cell proliferation
and viability.
The assay is based on the cleavage of the tetrazolium salt to
formazan by cellular mitochondrial dehydrogenase. The amount of
the dye generated by activity of dehydrogenase is directly
proportional to the number of living cells. The formazan dye
produced by viable cells can be quantified by multi-well
spectrophotometer (microtiter plate reader) by measuring the
absorbance of the dye solution at 440 nm.
The assay can be used for measurement of cell proliferation in
response to growth factors, cytokines, mitogens, and nutrients, etc. It
can also be used for the analysis of cytotoxic compounds like
anticancer drugs and many other toxic agents and pharmaceutical
compounds. The new method is so simple, just add-and-read,
requiring no washing, no harvesting, and no solubilization steps. It is
faster, stable, and more sensitive than MTT, XTT, MTS based
assays. The assay correlates well with the [3H]-thymidine
incorporation assay.
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2. Protocol Summary
Culture Cells
Add WST Solution
Incubate Cells
Shake Thoroughly
Measure Absorbance
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3. Components and Storage
A. Kit Components
Item Quantity Quantity
(500 assays) (2500 assays)
WST Reagent (Lyophilized) 1 vial 1 vial
Electro Coupling Solution (ECS) 5 ml 25 ml
Stop Solution 5 ml 25 ml
* Store kit at -20°C.
WST REAGENT: Dissolve the lyophilized WST reagent into
5 ml/25 ml Electro Coupling Solution (ECS), aliquot the solution (1 ml
is sufficient for one 96-well plate assay) and store at -20°C.
The WST Solution is stable for 1 year at -20°C and up to 6 months at
+4°C. Protect from light. Avoid repeated freeze-thaw. Repeated
freeze-thaw may increase background.
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B. Additional Materials Required
Microcentrifuge
Pipettes and pipette tips
Multi-well spectrophotometer (microtiter plate reader)
96 well plate
Orbital shaker
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4. Assay Protocol
1. Culture cells (0.1-5x104/well) in a 96-well microtiter plate in a final
volume of 100 μl/well culture medium in the absence or presence
of various amounts of the factors tested.
For toxicity assays, use more cells to start with (e.g., 5x104-x105
cells/well).
The optimal cell number used for the assay may vary among cell
types. For best results, it is recommended to add various
numbers of cells in your initial assay to determine the optimal cell
number and the developing time to be used.
2. Incubate cells for 24-96 hours.
3. Add 10 μl per well WST Solution to each well. Be careful not to
introduce bubbles to the wells.
4. Incubate the cells for 0.5-4 hours in standard culture conditions.
5. Shake thoroughly for 1 min. Measure the absorbance of the
treated and untreated samples using a microtiter plate reader at
420-480 nm according to the filters available for the plate reader.
The reference wavelength should be ~650nm.
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Notes:
a) Use the same amount of culture medium and WST reagent in an
empty well as a blank position for the microtiter plate reader.
b) The plate can be repeatedly read many times until OD reaches
1.8.
c) The reaction can be stopped by adding 10 μl of the Stop Solution
into each well, mix well, and the plate can be read within
48 hours. Protect from light and prevent medium evaporation.
d) Phenol Red in culture medium does not significantly interfere with
the assay.
e) WST shows very low toxicity and it does not stain the cells. Thus,
the same cells can be used for other tests after WST assay.
For further technical questions please do not hesitate to
contact us by email (
[email protected]) or phone (select
“contact us” on www.abcam.com for the phone number for
your region).
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UK, EU and ROW
Email:
[email protected]
Tel: +44 (0)1223 696000
www.abcam.com
US, Canada and Latin America
Email:
[email protected] Tel: 888-77-ABCAM (22226)
www.abcam.com
China and Asia Pacific
Email:
[email protected] Tel: 108008523689 (中國聯通)
www.abcam.cn
Japan
Email: [email protected]
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
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All information / detail is correct at time of going to print.
