Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria

Boris Zhivotosky1, Sten Orrenius1

1 Karolinska Institute, Stockholm, Sweden
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 18.3
DOI:  10.1002/0471143030.cb1803s12
Online Posting Date:  November, 2001
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Abstract

Apoptotic cells share a number of common features, such as phosphatidylserine (PS) exposure, cell shrinkage, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin. Necrotic cells exhibit nuclear swelling, chromatin flocculation, loss of nuclear basophilia, breakdown of cytoplasmic structure and organelle function, and cytolysis by swelling. This unit describes some of the techniques most commonly used to detect cell death. A number of assays are used for characterizing and distinguishing apoptosis and necrosis. Morphological assays include trypan blue exclusion, differential staining, and Hoechst staining. Methods to detect chromatin cleavage include TUNEL assays for whole cells and paraffin sections, DNA fragmentation assays using whole cells, assays of total genomic DNA, analysis of DNA fragmentation by agarose gel electrophoresis, phenol extraction of DNA for analysis of fragmentation, a quantitative assay for DNA fragmentation, and detection of DNA fragmentation by pulsedā€field gel electrophoresis. A protocol is also provided for Cytospin preparations from cell suspensions.

     
 
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Table of Contents

  • Morphology Assays
  • Basic Protocol 1: Measurement of Cell Death by Trypan Blue Exclusion
  • Basic Protocol 2: Differential Staining of Cells
  • Basic Protocol 3: Hoechst Staining of Cells
  • Support Protocol 1: Cytospin Preparation of Cells for Analysis
  • Assays for Chromatin Cleavage
  • Basic Protocol 4: TUNEL Assay for DNA Fragmentation in Cells
  • Alternate Protocol 1: TUNEL Assay in Paraffin‐Embedded Sections
  • Basic Protocol 5: Detection of DNA Fragmentation in Whole Cells
  • Alternate Protocol 2: Detection of DNA Fragmentation in Total Genomic DNA
  • Alternate Protocol 3: Simple Protocol for Detection of DNA Fragments
  • Alternate Protocol 4: Phenol Extraction of DNA Fragments for Agarose Gel Electrophoresis
  • Basic Protocol 6: Quantitative Assay of DNA Fragmentation
  • Basic Protocol 7: Detection of High‐Molecular‐Weight Chromatin Fragments by Pulsed‐Field Agarose Gel Electrophoresis
  • Methods for Analysis of Caspase Proteolytic Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Cell Death by Trypan Blue Exclusion

  Materials
  • Cell suspension to be assessed
  • 2× PBS tablets, pH 7.2 to 7.4 (Sigma) or 2× PBS ( appendix 2A)
  • 0.2% (w/v) trypan blue (Sigma) in 2× PBS (store up to 1 to 2 months at 4°C)
  • Hemacytometer: improved Neubauer type (Karl Hecht; Baxter) or equivalent
  • Coverslips (e.g., Chance Propper)
  • Light microscope

Basic Protocol 2: Differential Staining of Cells

  Materials
  • Cytospin preparations of cells (see protocol 4)
  • 100% methanol
  • Acid dye: 0.1% (w/v) eosin Y/0.1% (w/v) formaldehyde/0.4% (w/v) sodium phosphate dibasic/0.5% potassium phosphate monobasic
  • Basic dye: 0.4% (w/v) methylene blue‐polychromed/0.4% (w/v) azure/0.4% (w/v) sodium phosphate dibasic/0.5% (w/v) monobasic potassium phosphate
  • DPX mountant (in solution; BDH)
  • Coverslips (e.g., Chance Propper)
  • Microscope slides (e.g., Menzel‐Glaser)
  • Light microscope

Basic Protocol 3: Hoechst Staining of Cells

  Materials
  • Cell suspension
  • 1× and 2× PBS, pH 7.2 to 7.4 (from Sigma 2× PBS tablets, or see appendix 2A)
  • 4% (w/v) paraformaldehyde (see recipe)
  • 10 µg/ml Hoechst 33342 dye (Molecular Probes) in PBS ( appendix 2A)
  • 50/50 (v/v) PBS/glycerol
  • Microscope slides (e.g., Menzel‐Glaser)
  • Coverslips (e.g., Chance Propper)
  • Fluorescent microscope
  • Additional reagents and equipment for Cytospin preparations (see protocol 4)

Support Protocol 1: Cytospin Preparation of Cells for Analysis

  Materials
  • Cell suspension
  • PBS ( appendix 2A)
  • Cytospin centrifuge and cups (Shandon/Lipshaw)

Basic Protocol 4: TUNEL Assay for DNA Fragmentation in Cells

  Materials
  • Cell suspension
  • 100% methanol, −20°C
  • PBS, pH 7.2 to 7.4 ( appendix 2A)
  • 1% (v/v) formaldehyde
  • 70% ethanol, ice cold
  • Terminal deoxyribonucleotidyltransferase (TdT; 25 U/ml) and 10× buffer (0.3 M Tris base/1.4 M sodium cacodylate, pH 7.2/1 mM DTT; e.g., Boehringer Mannheim)
  • 25 mM CoCl 2
  • 1 mM Bio‐16‐dUTP (e.g., Boehringer Mannheim)
  • Termination buffer: 300 mM NaCl/30 mM sodium citrate (e.g., Sigma or equivalent)
  • Staining buffer (e.g., Sigma; see recipe)
  • FACS fluid (e.g., Becton Dickinson; optional; for flow cytometry)
  • DPX mountant (BDH)
  • 5 µg/ml propidium iodide in PBS ( appendix 2A)
  • Flow cytometer (e.g., Becton Dickinson) and tubes; or fluorescent microscope
  • Coverslips (e.g., Chance Propper)
  • Microscope slide (e.g., Objektträger, Menzel‐Glaser)

Alternate Protocol 1: TUNEL Assay in Paraffin‐Embedded Sections

  • Paraffin‐embedded tissue sections on slides
  • 4% (w/v) paraformaldehyde (see recipe) or 4% formaldehyde, in PBS ( appendix 2A)
  • 96%, 90%, and 80% ethanol
  • Xylene
  • BSA (e.g., Sigma)
  • 10 mM Tris⋅Cl, pH 8 ( appendix 2A)
  • 20 µg/ml proteinase K in 10 mM Tris⋅Cl ( appendix 2A for Tris⋅Cl)
  • 3% (v/v) methanol
  • 2% (w/v) BSA in PBS ( appendix 2A for PBS)
  • ExtrAvidin‐peroxidase (Sigma) diluted 1:50 in PBS/1% BSA/0.5% Tween 20
  • 3‐Amino‐9‐ethylcarbazole (AEC)
  • Fluorescent microscope
  • Coverslips (e.g., Chance Propper)
  • Microscope slide (e.g., Objektträger, Menzel‐Glaser)

Basic Protocol 5: Detection of DNA Fragmentation in Whole Cells

  Materials
  • 1× and 5× TBE buffer (see recipe), pH ∼8.0 at room temperature (do not adjust pH)
  • SeaKem GTG agarose (FMC Bioproducts)
  • Cell suspension
  • 50 mg/ml RNase A (see recipe)
  • 4× DNA loading buffer: 4× TBE buffer (see recipe) containing 40% (w/v) sucrose and 0.25% (w/v) bromphenol blue (e.g., Sigma; store up to 2 to 3 weeks at 4°C)
  • Ultrapure agarose (Life Technologies)
  • 10% (w/v) SDS (see recipe)
  • 20 mg/ml proteinase K in water (store in aliquots up to 1 year at −20°C)
  • 1× DNA loading buffer: 1× TBE buffer (see recipe) containing 10% (w/v) sucrose and 0.25% (w/v) bromphenol blue (e.g., Sigma)
  • DNA marker VI (pBR328 DNA cleaved with BglI and HinfI; Boehringer Mannheim), 1 µl in 20 µl of 1× DNA loading buffer
  • TE buffer, pH 8.0: 10 mM Tris⋅Cl/1 mM EDTA
  • 10 mg/ml ethidium bromide (see recipe)
  • Boiling water bath or microwave oven
  • Gel electrophoresis apparatus: GNA‐100 (Amersham Pharmacia Biotech), Buffer Puffer (Owl Scientific), or equivalent
  • Power supply (Power‐Pac 300, Bio‐Rad, or equivalent)
  • Shaker at 4°C
  • MacroVue UV Transilluminator (Hoefer Scientific Instruments) or equivalent
  • Photoman Polaroid gel documentation system (Hoefer Scientific Instruments) or equivalent

Alternate Protocol 2: Detection of DNA Fragmentation in Total Genomic DNA

  • Lysis buffer: 2 mM EDTA/100 mM Tris⋅Cl, pH 8.0/0.8% (w/v) SDS (store at room temperature)

Alternate Protocol 3: Simple Protocol for Detection of DNA Fragments

  • Lysis buffer, 4°C: 5 mM Tris⋅Cl, pH 8.0/20 mM EDTA/0.5% (v/v) Triton X‐100
  • 100% ethanol, −20°C
  • 5 M NaCl ( appendix 2A)
  • RNase T1/A stock (see recipe)
  • Vacuum lyophilizer (e.g., Hetovac, Heto‐Holten)

Alternate Protocol 4: Phenol Extraction of DNA Fragments for Agarose Gel Electrophoresis

  • Lysis buffer (see protocol 9), 4°C
  • 100% ethanol, −20°C
  • 5 M NaCl ( appendix 2A)
  • RNase T1/A stock (see recipe)
  • Phenol, TE‐saturated (see recipe)
  • 24:1 (v/v) chloroform/isoamyl alcohol (store mixture at room temperature in a fume hood)
  • 0.5% (w/v) SDS

Basic Protocol 6: Quantitative Assay of DNA Fragmentation

  Materials
  • Cell suspension to be assessed
  • Lysis buffer (see protocol 9), ice cold
  • 10% (w/v) and 5% (w/v) trichloroacetic acid (TCA; keep at room temperature in dark flasks)
  • Diphenylamine reagent (see recipe)
  • 10‐ml conical glass tubes
  • Round‐bottom glass tubes
  • Water bath, 100°C

Basic Protocol 7: Detection of High‐Molecular‐Weight Chromatin Fragments by Pulsed‐Field Agarose Gel Electrophoresis

  Materials
  • Cell suspension
  • Agarose buffer (for molds; see recipe)
  • SeaPlaque GTG low‐melting‐point agarose (FMC Bioproducts)
  • 20 mg/ml proteinase K in water (store in aliquots up to 1 year at −20°C)
  • Proteinase buffer (for plugs; see recipe)
  • TE buffer, pH 8.0:10 mM Tris⋅Cl/1 mM EDTA
  • 50 mM EDTA, pH 8.0 ( appendix 2A)
  • SeaKem GTG agarose (FMC Bioproducts)
  • 5× TBE buffer (see recipe)
  • DNA size pulse markers: chromosomes from Saccharomyces cerevisiae (225 to 2200 kbp) and a mixture of λDNA HindIII fragments, λDNA, and λDNA concatemers (0.1 to 200 kbp; Sigma; supplied premade in syringe)
  • Gel leveling table
  • 100‐µl insert molds (Amersham Pharmacia Biotech), stored in 0.1 M HCl
  • 12‐ or 24‐well tissue culture plates
  • 50°C incubator
  • 100°C water bath or microwave oven
  • Pulsed‐field gel electrophoresis system: vertical gel chamber with cooling elements (Protean II, Bio‐Rad); horizontal gel chamber (HE 100B); power supply (PS 500 XT); and Switchback pulse controller (PC 500, Hoefer Scientific Instruments)
  • Thermostatic circulator Multi Temp III (Amersham Pharmacia Biotech)
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Figures

Videos

Literature Cited

Literature Cited
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   Anand, R. and Southern, E.M. 1990. Pulsed field gel electrophoresis. In Gel Electrophoresis of Nucleic Acid: A Practical Approach, 2nd ed. (D. Rickwood and B.D. Hames, eds.) pp. 101‐123. IRL Press, Oxford.
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