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High‐Throughput Assessment of Mammalian Cell Viability by Determination of Adenosine Triphosphate Levels

Nicola Tolliday¹

¹Chemical Biology Platform, The Broad Institute of Harvard University and MIT, Cambridge, Massachusetts

Publication Name:

Current Protocols in Chemical Biology

Unit Number:

DOI:

10.1002/9780470559277.ch100045

Online Posting Date:

July, 2010

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Abstract

There are many assays available for high-throughput assessment of mammalian cell viability or cytotoxicity. Approaches include measurement of metabolic capacity, intracellular adenosine triphosphate (ATP) levels, induction of apoptosis, intracellular esterase and protease activity, and cellular membrane integrity. This unit provides an in-depth protocol for measurement of cellular ATP levels as a readout of mammalian cell viability, using the CellTiter-Glo assay from Promega Corporation. A comparison of the key parameters (sensitivity, speed, and cost) for this and other common high-throughput viability assays is also presented. Curr. Protoc. Chem. Biol. 2:153-161 © 2010 by John Wiley & Sons, Inc.

Keywords: CellTiter-Glo; cell viability assay; cytotoxicity assay; high-throughput screening

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Introduction
Basic Protocol: CellTiter-Glo Luminescent Cell Viability Assay
Support Protocol: Plating of NIH/3T3 Cells and Addition of Test Compounds by Pin Transfer
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol: CellTiter-Glo Luminescent Cell Viability Assay

Materials

CellTiter-Glo luminescent cell viability assay (Promega) containing:
- CellTiter-Glo buffer
- CellTiter-Glo substrate
NIH/3T3 cells seeded at 2500 cells/well in white opaque 384-well plates and treated with test compounds/perturbagens (for cell plating and compound treatment see the Support Protocol)

Vortex
Multichannel pipet or automated bulk dispenser/pipettor for reagent delivery to 384-well plates (see Rudnicki and Johnston, 2009)
Microtiter plate shaker/mixer
Luminometer or CCD imaging device capable of reading 384-well microtiter plates (for large numbers of plates, integration with a stacker or a robotic arm is helpful)

NOTE: Volumes given are for a 384-well plate. For a 96-well plate, scale up 4-fold. For a 1536-well plate, scale down 5-fold.

Support Protocol: Plating of NIH/3T3 Cells and Addition of Test Compounds by Pin Transfer

Materials

NIH/3T3 fibroblasts at ~70% to 80% confluence, grown in standard tissue culture flasks or plates using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with bovine calf serum to a final concentration of 10%
Phosphate-buffered saline (PBS; tissue culture grade)
0.5 mg/ml trypsin, 0.2 mg/ml ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS)
DMEM supplemented with bovine calf serum to a final concentration of 10%
Test compounds dissolved in DMSO (for example at 10 mM stock concentration) in 384-well polypropylene plates

Hemacytometer
Multichannel pipet or automated bulk dispenser/pipettor for reagent delivery to 384-well plates (see Rudnicki and Johnston, 2009)
White opaque 384-well microtiter plates (e.g., Corning, cat. no. 8867BC or similar)
37°C, 5% CO₂ incubator
Pin tool for delivery of test compounds into assay plates: both automated and manual options are available [see V&P Scientific link under Internet Resources for more information on both options; also see Rudnicki and Johnston (2009)]

Additional reagents and equipment for measuring cell viability (Basic Protocol)

Looking for materials? Suppliers Guide

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Figures

Figure 1.

The luciferase reaction. Mono-oxygenation of luciferin is catalyzed by luciferase in the presence of Mg²⁺, molecular oxygen, and ATP, resulting in the production of light (adapted from the Promega CellTiter-Glo manual).

View Image
Figure 2.

Representative data from the CellTiterGlo viability assay. Mammalian cells were incubated in 384-well plates for 3 days with Staurosporine ranging from 0.2 nM to 0.3 µM (16 replicates for each concentration). The effects on viability were determined using the CellTiterGlo assay, luminescence was read on an Envision Multilabel plate reader (PerkinElmer) and the data were analyzed using the curve fitting function within GraphPad Prism.

View Image

Literature Cited

Literature Cited
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	Boutros, M., Kiger, A.A., Armknecht, S., Kerr, K., Hild, M., Koch, B., Haas, S.A., Paro, R., Perrimon, N.; and Heidelberg Fly Array Consortium. 2004. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303:832-835.
	Crouch, S.P., Kozlowski, R., Slater, K.J., and Fletcher, J. 1993. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J. Immunol. Methods 160:81-88.
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	Rudnicki, S. and Johnston, S. 2009. Overview of liquid handling instrumentation for high-throughput screening applications. Curr. Protoc. Chem. Biol. 1:43-54.
	Scholl, C., Frohling, S., Dunn, I.F., Schinzel, A.C., Barbie, D.A., Kim, S.Y., Silver, S.J., Tamayo, P., Wadlow, R.C., Ramaswamy, S., Döhner, K., Bullinger, L., Sandy, P., Boehm, J.S., Root, D.E., Jacks, T., Hahn, W.C., and Gilliland, D.G. 2009. Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell 137:821-834.
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Internet Resources
	http://www.promega.com/paguide/chap4.htm
A protocols and application guide from Promega that provides an overview to cell viability and cytotoxicity assays.
	http://www.promega.com/catalog/catalogproducts.aspx categoryname=productleaf_1505
The CellTiter-Glo technical manual from Promega.
	http://www.vp-scientific.com/pin_tools.htm
Information on manual and automated pin tools for delivery of compounds into assay plates.
	http://www.labautopedia.org/mw/index.php/User:S.d.hamilton/Helpful_Hints...
Helpful Hints to Manage Edge Effect of Cultured Cells for High Throughput Screening: A Corning HTS/Assay Systems Application Note that addresses common causes of edge effects observed with cell-based assays, authored by Allison Tanner.