Morphological, Biochemical, and Flow Cytometric Assays of Apoptosis

David Martin1, Michael Lenardo1

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.13
DOI:  10.1002/0471142727.mb1413s49
Online Posting Date:  May, 2001
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Abstract

As programmed cell death (PCD), or apoptosis, has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. Necrosis refers to the morphology usually associated with accidental cell death, while apoptosis is seen when cell death is programmed or physiologically regulated. This unit presents a set of assays for these purposes, many of which are technically very simple. Featured in this unit is the TUNEL method of detecting cells that exhibit DNA fragmentation, which can also be performed on tissue sections to locate apoptotic cells in situ.

     
 
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Table of Contents

  • Basic Protocol 1: Microscopic Quantitation of Apoptotic Index and Cell Viability Using Vital and Fluorescent Dyes
  • Basic Protocol 2: Determination of Apoptosis Using Sub‐G0/G1 DNA Peak
  • Basic Protocol 3: Flow Cytometric Quantitation of Apoptotic Cells Using TUNEL
  • Basic Protocol 4: In Situ Detection of Apoptotic Cells in Tissue Sections by TUNEL
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microscopic Quantitation of Apoptotic Index and Cell Viability Using Vital and Fluorescent Dyes

  Materials
  • Dye mix: 0.01% trypan blue (Life Technologies), 100 µg/ml acridine orange (Sigma), or 100 µg/ml acridine orange + 100 µg/ml ethidium bromide (Sigma), all prepared in PBS ( appendix 22)
  • Cell suspension at ∼5 × 105 to 5 × 106 cells/ml (Table 14.13.1) in complete RPMI 1640 medium ( appendix 3F)
    Table 4.3.1   Materials   Stimulation of Apoptosis in Cultured Cells a   Stimulation of Apoptosis in Cultured Cells

    Cell type Treatment Assay time (hr) b Apoptotic index c Protein synthesis required d
    Thymocytes 100 nM dexamethasone600 rad γ‐irradiation 2424 80‐10080‐100 YesYes
    D011.10 T hybridoma Anti‐CD3 24 80 Yes
    P815 mastocytoma Cytotoxic T cells 4 50‐100 No
    Raji B cells Cytotoxic T cells 4 10‐20 No

     aSee Background Information for a further description of the cell types and treatments employed in these studies.
     bApproximate time until the cells are assessed for apoptosis.
     cApoptotic index refers to the expected values for the percent of apoptotic/dead cells at the indicated times as quantified according to protocol 1.
     dRequirement of protein synthesis refers to whether inhibitors of RNA (e.g., actinomycin D) or protein (e.g., cycloheximide, emetine, and pactamycin) synthesis prevent apoptosis (nuclear damage as well as cell lysis).
  • 12 × 75–mm glass tube
  • Microscope slide and 22‐mm2 coverslip (no. 1 thickness; VWR Scientific)
  • Fluorescence microscope (e.g., Zeiss or equivalent) equipped with a fluorescein filter set
  • Additional reagents and equipment for counting cells ( appendix 3F)

Basic Protocol 2: Determination of Apoptosis Using Sub‐G0/G1 DNA Peak

  Materials
  • Cells to be analyzed, in recipeFACS buffer (see recipe; 105 to 106 total)
  • recipeFACS buffer (see recipe)
  • Ice‐cold 100% ethanol (70% if cells are to be antibody‐labeled)
  • 1 mg/ml RNase A
  • 50 µg/ml propidium iodide (PI) in PBS ( appendix 22)

Basic Protocol 3: Flow Cytometric Quantitation of Apoptotic Cells Using TUNEL

  Materials
  • Cells for analysis
  • PBS ( appendix 22)
  • 95% ethanol (not 100% ethanol), ice cold
  • recipeParaformaldehyde fixative solution (see recipe)
  • recipeTdT reaction buffer (see recipe)
  • recipeTdT/biotin‐dUTP mix (see recipe)
  • Fluorescein isothiocyanate (FITC)‐conjugated streptavidin (Jackson Immunoresearch; follow manufacturer's instructions for appropriate dilution)
  • 12 × 75–mm round‐bottom centrifuge tubes
  • IEC 6R6000 centrifuge with model 269 rotor (or equivalent)
  • Additional reagents and equipment for immunofluorescence staining (optional; Holmes and Fowlkes, ) and flow cytometric analysis (Otten et al., )

Basic Protocol 4: In Situ Detection of Apoptotic Cells in Tissue Sections by TUNEL

  Materials
  • Fresh tissues for analysis
  • 1% (w/v) paraformaldehyde in PBS (dissolve by stirring with low heat overnight and filter before use)
  • Tris‐buffered saline (TBS; appendix 22)
  • 0.1% (v/v) H 2O 2 in TBS
  • recipeTdT reaction buffer (see recipe)
  • recipeTdT/digoxigenin‐dUTP mix (see recipe)
  • 2% (v/v) horse serum or FBS in TBS
  • recipeSheep anti‐digoxigenin primary antibody solution (see recipe)
  • recipeHRPO‐conjugated anti‐sheep secondary antibody solution (see recipe)
  • recipeAEC substrate working solution (see recipe)
  • Mayer's hematoxylin (Sigma)
  • Crystal Mount mounting medium (Fisher)
  • Hydrophobic‐barrier slide marker (e.g., PAP Pen; Research Products International)
  • Coplin jars or staining trays
  • recipeHumidified container (see recipe)
  • Additional reagents and equipment for preparing frozen sections (as in immunoperoxidase staining; unit 14.2 & )
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Figures

Videos

Literature Cited

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Key Reference
   Kerr et al., 1972. See above.
  Generalizes the distinction between programmed and pathological cell death based on differences in morphology and suggests the term apoptosis, derived from Greek and meaning “falling off,” as of leaves from trees.
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