Multiparameter Analysis of Physiological Changes in Apoptosis

Martin Poot1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.15
DOI:  10.1002/0471142956.cy0915s14
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Changes in mitochondrial parameters leading to cells with compromised mitochondrial function are a hallmark of apoptosis. These changes can be detected and the number of compromised cells quantitated by multicolor flow cytometry utilizing fluorescent dyes that monitor specific features of this organelle. In this way one can trace physiological changes during apoptosis and investigate whether or not certain changes occur simultaneously in the same cell. This unit presents protocols for combined NADH levels and mitochondrial membrane potential, cell cycle stage specific apoptosis, and cellular thiol and mitochondrial cardiolipin levels.

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

Table of Contents

  • Basic Protocol 1: Combined Assay for NADPH Levels and Mitochondrial Membrane Potential
  • Basic Protocol 2: Assay for Cell Cycle Stage–Specific Apoptosis
  • Basic Protocol 3: Combined Assay for Cellular Reduced Thiol and Cardiolipin Levels
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Combined Assay for NADPH Levels and Mitochondrial Membrane Potential

  Materials
  • Cultured cells ( appendix 3B)
  • Culture medium (optimal for cell‐type), 37°C
  • 200 µM stock solutions of MitoTracker Green FM and CMXRosamine in DMSO; store at −20°C in the dark
  • 15‐ml screw‐capped centrifuge tubes
  • 37°C water bath with cover (e.g., a sheet of aluminum)
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer with either a mercury arc lamp or two argon lasers (tuned to ultraviolet and to 488 nm) as excitation source
  • Computer for data collection and processing

Basic Protocol 2: Assay for Cell Cycle Stage–Specific Apoptosis

  Materials
  • Cells in suspension (see protocol 1, step )
  • 200 µM stock solutions of MitoTracker Green FM and CMXRosamine in DMSO; store at −20°C in the dark
  • 1 mM stock solution of Hoechst 33342 dye in double‐distilled water; store at 4°C in the dark (do not freeze)
  • 12 × 75–mm polypropylene tubes
  • 37°C water bath with cover (e.g., a sheet of aluminum)
  • Flow cytometer with either a mercury arc lamp or two argon lasers (tuned to ultraviolet and to 488 nm) as excitation source
  • Computer for data collection and processing
NOTE: Do not use phosphate‐containing buffers, since Hoechst dyes precipitate with phosphates.

Basic Protocol 3: Combined Assay for Cellular Reduced Thiol and Cardiolipin Levels

  Materials
  • Cells in suspension (see protocol 1, step )
  • 10 mM stock solution of monobromobimane in absolute ethanol; store at 4°C in the dark
  • 1 mM stock solution of nonyl acridine orange (NAO) in absolute ethanol; store at 4°C in the dark
  • 12 × 75–mm polypropylene tubes
  • 37°C water bath with cover (e.g., a sheet of aluminum)
  • Flow cytometer with either a mercury arc lamp or two argon lasers (tuned to ultraviolet and to 488 nm) as excitation source
  • Computer for data collection and processing
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Backway, K.L., McCulloch, E.A., Chow, S., and Hedley, D.W. 1997. Relationships between the mitochondrial permeability transition and oxidative stress during ara‐C toxicity. Cancer Res. 57:2446‐2451.
   Cossarizza, A., Kalashnikova, G., Grassilli, E., Chiappelli, F., Salvioli, S., Capri, M., Barbieri, D., Troiano, L., Monti, D., and Franceschi, C. 1994. Mitochondrial modifications during rat thymocyte apoptosis: A study at the single cell level. Exp. Cell Res. 214:323‐330.
   Gallet, P.F., Maftah, A., Petit, J.M., Denis‐Gay, M., and Julien, R. 1995. Direct cardiolipin assay in yeast using the red fluorescence emission of 10‐N‐nonyl acridine orange. Eur. J. Biochem. 228:113‐119.
   Green, D. and Kroemer, G. 1998. The central executioners of apoptosis: Caspases or mitochondria? Trends Cell. Biol. 8:267‐271.
   Green, D.R. and Reed, J.C. 1998. Mitochondria and apoptosis. Science 281:1309‐1312.
   Macho, A., Decaudin, D., Castedo, M., Hirsch, T., Susin, S.A., Zamzami, N., and Kroemer, G. 1996. Chloromethyl‐X‐Rosamine is an aldehyde‐fixable potential‐sensitive fluorochrome for the detection of early apoptosis. Cytometry 25:333‐340.
   Poot, M. and Pierce, R.H. 1999. Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry. Cytometry 35:311‐317.
   Poot, M., Verkerk, A., Koster, J.F., and Jongkind, J.F. 1986. De novo synthesis of glutathione in human fibroblasts during in vitro aging and in some metabolic diseases as measured by a flow cytometric method. Biochim. Biophys. Acta. 883:580‐584.
   Thorell, B. 1983. Flow‐cytometric monitoring of intracellular flavins simultaneously with NAD(P)H levels. Cytometry 4:61‐65.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library