Assessment of Viability, Immunofluorescence, and DNA Content

Ingrid Schmid1

1 UCLA School of Medicine, Los Angeles, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.11
DOI:  10.1002/0471142956.cy0711s10
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit addresses the difficult and important area of differentiation between viable and nonviable cells, a question of particular interest for DNA studies. The basic protocol describes the determination of DNA content using pyronin Y combined with dead cell discrimination using 7‐AAD. Alternate protocols present methods for DNA content and viability assessment and simultaneous surface or intracellular antigen expression.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: DNA Content Analysis in Combination with Assessment of Cell Viability
  • Alternate Protocol 1: Analysis of DNA Content and Cell‐Surface Antigen Expression Combination with Assessment of Cell Viability
  • Alternate Protocol 2: Analysis of DNA Content and Intracellular Antigen Expression in Combination with Assessment of Cell Viability
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: DNA Content Analysis in Combination with Assessment of Cell Viability

  Materials
  • Cell suspension (e.g., cells from suspension cultures; Ficoll‐Hypaque‐purified cells, either mononuclear cell fraction or suspension of cells isolated from tissue)
  • PBS ( appendix 2A)
  • 1 mg/ml 7‐aminoactinomycin D (7‐AAD) stock solution (see recipe)
  • Staining buffer (see recipe)
  • 1 mg/ml actinomycin D (AD) stock solution (see recipe)
  • Fixation solution (see recipe)
  • Permeabilization solution (see recipe), 37°C
  • DNase‐free RNase A (Sigma)
  • 100 µg/ml pyronin Y (PY; also called pyronin G; Polysciences) stock solution in deionized, distilled H 2O
  • 12 × 75–mm culture tubes
  • 37°C water bath
  • Flow cytometer with 488‐nm excitation and appropriate collection filters for PY (585‐nm band‐pass) and 7‐AAD (650‐ or 670‐nm long‐pass)
  • Additional reagents and equipment for counting cells ( appendix 3A)

Alternate Protocol 1: Analysis of DNA Content and Cell‐Surface Antigen Expression Combination with Assessment of Cell Viability

  • FITC‐labeled antibody to antigen of interest
  • FITC‐labeled isotypic control antibody

Alternate Protocol 2: Analysis of DNA Content and Intracellular Antigen Expression in Combination with Assessment of Cell Viability

  • FITC‐labeled antibody to antigen of interest
  • FITC‐labeled isotypic control antibody
  • Wash solution (see recipe)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Boltz, R.C., Fischer, P.A., Wicker, L.S., and Peterson, L.B. 1994. Single UV excitation of Hoechst 33342 and ethidium bromide for simultaneous cell cycle analysis and viability determinations on in vitro cultures of murine B lymphocytes. Cytometry 15:28‐34.
   Darzynkiewicz, Z., Kapuscinski, J., Traganos, F., and Crissman, H.A. 1987. Application of pyronin Y(G) in cytochemistry of nucleic acids. Cytometry 8:138‐145.
   Fetterhoff, T.J., Holland, S.P., and Wile, K.J. 1993. Fluorescent detection of non‐viable cells in fixed cell preparations [Abstract]. Cytometry Supplement 6:27.
   Kapuscinski, J. and Darzynkiewicz, Z. 1987. Interactions of pyronin Y(G) with nucleic acids. Cytometry 8:129‐137.
   Muirhead, K.A., Kloszewski, E.D., Antell, L.A., and Griswold, D.E. 1985. Identification of live cells for flow cytometric analysis of lymphoid subset proliferation in low viability populations. J. Immunol. Methods 77:77‐86.
   Müller, W. and Crothers, D.M. 1975. Interactions of heteroaromatic compounds with nucleic acids. Eur. J. Biochem. 54:267‐277.
   Schmid, I. and Giorgi, J.V. 1995. Section on Intracellular Staining In Holmes, K., Foulkes, B.J., Schmid, I., and Giorgi, J.V. Preparation of cells and reagents for flow cytometry. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 5.3.1‐5.3.23. John Wiley & Sons, New York.
   Schmid, I., Uittenbogaart, C.H., and Giorgi, J.V. 1991. A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification. Cytometry 12:279‐285.
   Schmid, I., Ferbas, J., Uittenbogaart, C.H., and Giorgi, J.V. 1999. Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability. Cytometry 35:64‐74.
   Shapiro, H.M. 1981. Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and Pyronin Y. Cytometry 2:143‐150.
   Tanke, H.J., Nieuwenhuis, I.A.B., Koper, G.J.M., Slats, J.C.M., and Ploem, J.S. 1980. Flow cytometry of human reticulocytes based on RNA fluorescence. Cytometry 1:313‐320.
   Toba, K., Winton, E.F., Koike, T., and Shibata, A. 1995. Simultaneous three‐color analysis of the surface phenotype and DNA‐RNA quantitation using 7‐aminoactinomycin D and pyronin Y. J. Immunol. Methods 182:193‐207.
Key Reference
   Schmid et al., 1999. See above.
  Describes the procedures presented in the and Alternate Protocols 1 and 2.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library