Trypan Blue Exclusion Test of Cell Viability

Warren Strober1

1 National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 3B
DOI:  10.1002/0471142735.ima03bs111
Online Posting Date:  November, 2015
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Abstract

The protocol described in this appendix allows for light microscopic quantitation of cell viability. Cells are suspended in PBS containing trypan blue and then examined to determine the percentage of cells that have clear cytoplasm (viable cells) versus cells that have blue cytoplasm (nonviable cells). © 2015 by John Wiley & Sons, Inc.

Keywords: trypan blue; cell viability; dye exclusion

     
 
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Table of Contents

  • Basic Protocol 1:  
  • Commentary
     
 
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Materials

Basic Protocol 1:  

  Materials
  • Cell suspension to be tested for viability
  • PBS ( ) or serum‐free complete medium ( )
  • 0.4% trypan blue (store in dark bottle and filter after prolonged storage; Life Technologies)
  • Hemacytometer ( ; Strober, )
  • Microtiter plate or small plastic tube
  • Binocular microscope
  • Additional reagents and equipment for counting cells using a hemacytometer ( ; Strober, )
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Figures

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Literature Cited

Literature Cited
   Avelar‐Freitas, B.A. , Almeida, V.G. , Pinto, M.C.X. , Mourao, F.A.G. , Massensini, A.R. , Martins‐Filo, O.A. , Rocha‐Vieira, E. , and Brito‐Melo, G.E.A. 2014. Trypan blue exclusion assay by flow cytometry. Brazilian J. Med. Biol. Res. 47:307‐315. http://dx.doi.org/10.1590%2F1414‐431X20143437
   Holmes, K. L. , Otten, G. and Yokoyama, W.M. 2002. Flow cytometry analysis using the Becton Dickinson FACS Calibur. Curr. Protoc. Immunol. 49:5.4.1‐5.4.22.
   Kim, J.S. , Nam, M.H. , An, S.A. , Lim, C.S. , Hur, D.S. Chung, C. , and Chang, J.K. 2011. Comparison of the automated fluorescence microscopic viability test with conventional and flow cytometry methods. J. Clin. Lab. Anal. 25:90‐94. doi: 10.1002/jcla.20438.
   Strober, W. 1997. Monitoring cell growth. Curr. Protoc. Immunol. 21:A.3A.1‐A.3A.2.
Key References
   Avelar‐Freitas et al., 2014. See above.
  This article provides a comprehensive survey of methods of measuring cell viability by dye exclusion.
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