Measuring Apoptosis by Microscopy and Flow Cytometry

Emilie Hollville1, Seamus J. Martin1

1 Molecular Cell Biology Laboratory, Department of Genetics, Trinity College, Dublin
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.38
DOI:  10.1002/0471142735.im1438s112
Online Posting Date:  February, 2016
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Abstract

Apoptosis is a mode of programmed cell death that plays an important role during development and in the maintenance of tissue homeostasis. Numerous physiological as well as pathological stimuli trigger apoptosis such as engagement of Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, or exposure to cytotoxic drugs. Apoptosis is coordinated from within by members of the caspase family of cysteine proteases that, upon activation, trigger a series of morphological changes including cell shrinkage, extensive plasma membrane blebbing, chromatin condensation, DNA hydrolysis, and nuclear fragmentation. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the efficient recognition and removal of apoptotic cells by phagocytes. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by rapid plasma membrane rupture followed by organelle and cell swelling. Necrosis is often provoked by infectious agents or a severe departure from physiological conditions. This unit describes protocols for the measurement of apoptosis and for distinguishing apoptosis from necrosis. © 2016 by John Wiley & Sons, Inc.

Keywords: apoptosis; necrosis; cell morphology; microscopy; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Cell Morphology by Phase‐Contrast Microscopy
  • Alternate Protocol 1: Assessment of Morphological Changes Using Eosin‐Methylene Blue Staining
  • Alternate Protocol 2: Analysis of Nuclear Morphology by Fluorescence Microscopy
  • Support Protocol 1: Preparation of Cytospins
  • Basic Protocol 2: Measurement of Plasma Membrane Composition with Annexin V and Propidium Iodide
  • Basic Protocol 3: Measurement of DNA Fragmentation by Flow Cytometry
  • Alternate Protocol 3: Analysis of DNA Fragmentation by the TUNEL Assay
  • Basic Protocol 4: Measurement of Caspase Activation by Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Analysis of Cell Morphology by Phase‐Contrast Microscopy

  Materials
  • Cells of interest
  • Phase‐contrast microscope
  • High‐resolution camera (optional)
  • Hemacytometer (optional)

Alternate Protocol 1: Assessment of Morphological Changes Using Eosin‐Methylene Blue Staining

  Materials
  • Cytospins (see protocol 4Support Protocol)
  • 100% methanol
  • 1% (w/v) Eosin Y staining solution (see recipe)
  • 1% (w/v) methylene blue staining solution (see recipe)
  • DPX mounting medium
  • Coplin staining jar
  • Coverslips, No. 1 thickness
  • Phase‐contrast microscope equipped with a high‐resolution, color camera

Alternate Protocol 2: Analysis of Nuclear Morphology by Fluorescence Microscopy

  Materials
  • Cells grown on coverslips (No. 1 thickness) or cytospins (see protocol 4Support Protocol)
  • PBS (see appendix 2A)
  • 3.5% (w/v) paraformaldehyde (see recipe)
  • 0.15% (v/v) Triton X‐100 (see recipe)
  • 2 μg/ml Hoechst 33342 staining solution (see recipe)
  • Antifade fluorescence mounting medium (e.g., SlowFade or equivalent)
  • Clear nail varnish
  • Fluorescence (or confocal) microscope with a xenon or mercury lamp and a blue filter

Support Protocol 1: Preparation of Cytospins

  Materials
  • Single‐cell suspension sample
  • PBS (see appendix 2A)
  • Hemacytometer
  • Centrifuge
  • Cytocentrifuge (e.g., ThermoShandon)
  • Cytospin slides (e.g., Sigma)
  • Cytospin filters (e.g., ThermoShandon)
  • Cytospin chambers (e.g., ThermoShandon)

Basic Protocol 2: Measurement of Plasma Membrane Composition with Annexin V and Propidium Iodide

  Materials
  • Single‐cell suspension at a density of 106 cells/ml
  • 1 μg/ml annexin V‐FITC staining solution (see recipe)
  • 1 mg/ml propidium iodide (PI) stock solution (see recipe)
  • Flow cytometry tubes
  • Centrifuge
  • Flow cytometer with a 488 nm argon ion laser

Basic Protocol 3: Measurement of DNA Fragmentation by Flow Cytometry

  Materials
  • Single‐cell suspension
  • PBS (see appendix 2A)
  • 100% ethanol, ice cold
  • Phosphate‐citrate wash buffer (see recipe)
  • 50 μg/ml propidium iodide (PI) staining solution containing 100 μg/ml DNase‐free RNase A (see recipe)
  • Centrifuge
  • 1.5‐ml microcentrifuge tubes
  • Ice
  • Flow cytometry tubes
  • Flow cytometer with a 488 nm argon ion laser

Alternate Protocol 3: Analysis of DNA Fragmentation by the TUNEL Assay

  Materials
  • Single‐cell suspension or cells grown on coverslips (No. 1 thickness)
  • PBS (see appendix 2A)
  • 3.5% (w/v) paraformaldehyde (see recipe)
  • 0.15% (v/v) Triton X‐100 (see recipe)
  • TdT equilibration buffer (see recipe)
  • TdT reaction solution (see recipe)
  • FITC‐conjugated anti‐BrdU staining solution (see recipe)
  • Centrifuge
  • 1.5‐ml microcentrifuge tubes
  • Ice
  • 37°C incubator
  • Flow cytometer with a 488 nm argon ion laser

Basic Protocol 4: Measurement of Caspase Activation by Flow Cytometry

  Materials
  • Single‐cell suspension
  • Cell culture medium appropriate for the cells of interest
  • 300 μM FAM‐VAD‐FMK FLICA reagent (e.g., Molecular Probes or ImmunoChemistry Technologies) in PBS (see appendix 2A)
  • 1 mg/ml propidium iodide (PI) stock solution (see recipe)
  • Centrifuge
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator
  • Flow cytometer with a 488 nm argon ion laser
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Figures

Videos

Literature Cited

Literature Cited
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