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

Nicola Tolliday1

1 Chemical 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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Table of Contents

  • Introduction
  • Basic Protocol 1: CellTiter‐Glo Luminescent Cell Viability Assay
  • Support Protocol 1: 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 1: 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 protocol 2)
  • Vortex
  • Multichannel pipet or automated bulk dispenser/pipettor for reagent delivery to 384‐well plates (see Rudnicki and Johnston, )
  • 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 1: 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, )
  • White opaque 384‐well microtiter plates (e.g., Corning, cat. no. 8867BC or similar)
  • 37°C, 5% CO 2 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 ( )]
  • Additional reagents and equipment for measuring cell viability ( protocol 1)
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Figures

Videos

Literature Cited

   Barbie, D.A., Tamayo, P., Boehm, J.S., Kim, S.Y., Moody, S.E., Dunn, I.F., Schinzel, A.C., Sandy, P., Meylan, E., Scholl, C., Fröhling, S., Chan, E.M., Sos, M.L., Michel, K., Mermel, C., Silver, S.J., Weir, B.A., Reiling, J.H., Sheng, Q., Gupta, P.B., Wadlow, R.C., Le, H., Hoersch, S., Wittner, B.S., Ramaswamy, S., Livingston, D.M., Sabatini, D.M., Meyerson, M., Thomas, R.K., Lander, E.S., Mesirov, J.P., Root, D.E., Gilliland, D.G., Jacks, T., and Hahn, W.C. 2009. Systematic RNA interference reveals that oncogenic KRAS‐driven cancers require TBK1. Nature 462:108‐112.
   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.
   Firestein, R., Bass, A.J., Kim, S.Y., Dunn, I.F., Silver, S.J., Guney, I., Freed, E., Ligon, A.H., Vena, N., Ogino, S., Chheda, M.G., Tamayo, P., Finn, S., Shrestha, Y., Boehm, J.S., Jain, S., Bojarksi, E., Mermel, C., Barretina, J., Chan, J.A., Baselga, J., Tabernero, J., Root, D.E., Fuchs, C.S., Loda, M., Shivdasani, R.A., Meyerson, M., and Hahn, W.C. 2008. CDK8 is a colorectal cancer oncogene that regulates beta‐catenin activity. Nature 455:547‐551.
   Freshney, I.R. 2005. Culture of Animal Cells: A Manual of Basic Technique, 5th ed. Wiley‐Liss, Hoboken, N.J.
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   Gupta, P.B., Onder, T.T., Jiang, G., Tao, K., Kuperwasser, C., Weinberg, R.A., and Lander, E.S. 2009. Identification of selective inhibitors of cancer stem cells by high‐throughput screening. Cell 138:645‐659.
   Hahn, C.K., Ross, K.N., Warrington, I.M., Mazitschek, R., Kanegai, C.M., Wright, R.D., Kung, A.L., Golub, T.R., and Stegmaier, K. 2008. Expression‐based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation. Proc. Natl. Acad. Sci. U.S.A. 105:9751‐9756.
   Lightfield, K.L., Persson, J., Brubaker, S.W., Witte, C.E., von Moltke, J., Dunipace, E.A., Henry, T., Sun, Y., CadoD., Dietrich, W.F., Monack, D.M., Tsolis, R.M., and Vance, R.E. 2008. Critical function for Naip5 in inflammasome activation by a conserved carboxy‐terminal domain of flagellin. Nat. Immunol. 9:1171‐1178.
   Lundholt, B.K., Scudder, K.M., and Pagliaro, L. 2003. A simple technique for reducing edge effect in cell‐based assays. J. Biomol. Screen. 8:566‐570.
   Piantadosi, C.A. and Suliman, H.B. 2006. Mitochondrial transcription factor A induction by redox activation of nuclear respiratory factor 1. J. Biol. Chem. 281:324‐333.
   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.
   Wagner, B.K., Kitami, T., Gilbert, T.A., Peck, D., Ramanathan, A., Schreiber, S.L., Golub, T., and Mootha, V. 2008. Large‐scale chemical dissection of mitochondrial function. Nat. Biotechnol. 26:343‐51.
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_to_Manage_Edge_Effect_of_Cultured_Cells_for_High_Throughput_Screening
  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.
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