Determination of Apoptosis and Necrosis

Boris Zhivotovsky1, Afshin Samali1, Sten Orrenius1

1 Karolinska Institute, Stockholm
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.2
DOI:  10.1002/0471140856.tx0202s00
Online Posting Date:  May, 2001
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Abstract

Cell death occurs by one of two mechanisms: apoptosis (programmed cell death) or necrosis due to cellular injury or trauma. Each method has distinct morphological and biochemical characteristics. Distinguishing between apoptosis and necrosis depends selection of appropriate assays and proper timing. This unit provides an extensive series of assays that measure membrane integrity, cell size and DNA content, state of chromatin, DNA fragmentation, and caspase activation. The results of these assays allow one to distinguish between apoptosis and necrosis.

     
 
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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
  • Basic Protocol 4: Flow Cytometric Analysis to Detect Apoptotic Cells
  • Basic Protocol 5: Assessment of Annexin V Binding to Detect Externalized Phosphatidylserine and Propidium Iodide Exclusion
  • Assays for Chromatin Cleavage
  • Basic Protocol 6: TUNEL Assay for DNA Fragmentation in Cells
  • Alternate Protocol 1: TUNEL Assay in Paraffin‐Embedded Sections
  • Basic Protocol 7: 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 8: Quantitative Assay of DNA Fragmentation
  • Basic Protocol 9: Detection of High‐Molecular‐Weight Chromatin Fragments by Pulsed‐Field Agarose Gel Electrophoresis
  • Methods for Analysis of Caspase Proteolytic Activity
  • Basic Protocol 10: Detection of Caspase Activity by Specific Substrate Cleavage
  • Basic Protocol 11: Immunodetection of Active Caspases During Apoptosis
  • Support Protocol 2: Preparation of SDS‐Polyacrylamide Minigels
  • Reagents and Solutions
  • Commentary
  • 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: Flow Cytometric Analysis to Detect Apoptotic Cells

  Materials
  • Cell suspension
  • 70% ethanol, ice cold
  • 1× PBS, pH 7.2 to 7.4 ( appendix 2A)
  • 50 µg/ml propidium iodide (Sigma) in 1× PBS ( appendix 2A for PBS; store up to 1 month at 4°C)
  • 25 mg/ml RNase A (e.g., Sigma) in water
  • Flow cytometer (e.g., Becton Dickinson) and tubes

Basic Protocol 5: Assessment of Annexin V Binding to Detect Externalized Phosphatidylserine and Propidium Iodide Exclusion

  Materials
  • Cell suspension
  • PBS, pH 7.2 to 7.4 ( appendix 2A)
  • 1 to 3 µg/ml annexin V–FITC (Bender Biosystems, Nexin Research, Roche, or Pharmingen)
  • HEPES buffer, pH 7.4 (see recipe)
  • HEPES buffer (see recipe) containing 50 µg/ml propidium iodide (e.g., Sigma)
  • Flow cytometer (e.g., Becton Dickinson) and tubes

Basic Protocol 6: 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/µl) 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 7: 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: recipe2 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/ recipe20 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 11), 4°C
  • 100% ethanol, −20°C
  • 5 M NaCl ( appendix 2A)
  • RNase T1/A stock (see recipe)
  • TE‐saturated phenol (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 8: Quantitative Assay of DNA Fragmentation

  Materials
  • Cell suspension to be assessed
  • Lysis buffer (see protocol 11), 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 9: 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)

Basic Protocol 10: Detection of Caspase Activity by Specific Substrate Cleavage

  Materials
  • Cell suspension
  • 1× PBS, pH 7.2 to 7.4 ( appendix 2A)
  • 1× substrate buffer (SB; see recipe)
  • Caspase substrate and inhibitor stock solutions (see recipe)
  • Liquid nitrogen
  • 96‐well tissue culture plates (e.g., LabSystems)
  • Fluoroscan II (LabSystems)

Basic Protocol 11: Immunodetection of Active Caspases During Apoptosis

  Materials
  • Cell suspension
  • PBS, pH 7.2 to 7.4 ( appendix 2A)
  • Sample buffer (see recipe)
  • Prestained SDS‐PAGE markers, low range (e.g., Bio‐Rad)
  • 1× electrode buffer (see recipe)
  • 4× high‐salt Tris base buffer (HSB; see recipe)
  • High‐salt Tris base buffer with Tween 20 (HSBT; see recipe)
  • Low‐salt Tris buffer (LSB; see recipe)
  • Transfer buffer (see recipe)
  • Dry nonfat milk
  • BSA fraction V (e.g., Sigma)
  • Sodium azide
  • Stripping buffer (see recipe)
  • Primary antibodies:
  •  Anti–caspase 1 (Calbiochem)
  •  Anti–caspase 2 (Transduction Laboratories)
  •  Anti–caspase 3 (Pharmingen)
  •  Anti–caspase 6 (Research Diagnostics)
  •  Anti–caspase 7 (Transduction Laboratories)
  •  Anti–caspase 8 (Pharmingen)
  •  Anti–caspase 9 (Calbiochem)
  •  Anti–caspase 10 (Calbiochem)
  • Appropriate secondary antibodies for the primary antibodies listed above: peroxidase‐conjugated goat anti–rabbit IgG, goat anti–mouse IgG, and mouse anti–goat IgG (Pierce)
  • Minigel (see protocol 17)
  • Anti‐glyceraldehyde‐3‐phosphate dehydrogenase (Trevigen)
  • Anti‐actin (Sigma or Boehringer Mannheim)
  • ECL Western blotting detection system (Amersham Pharmacia Biotech) or equivalent
  • Boiling water bath
  • Mini‐Protean II Electrophoresis Cell (Bio‐Rad) or equivalent
  • Prot/Elec pipet tips for protein gel (Bio‐Rad)
  • Power supply (Power‐Pac 300, Bio‐Rad, or equivalent)
  • 0.45‐µM nitrocellulose membranes (Sartorius AG)
  • Whatman 3MM filter paper
  • Mini Trans‐Blot Transfer Cell (Bio‐Rad) or equivalent
  • X‐ray film (e.g., FujiFilm)
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE, minigels (see protocol 17), and immunoblotting (unit 2.3)

Support Protocol 2: Preparation of SDS‐Polyacrylamide Minigels

  Materials
  • 29% (w/v) acrylamide/1% (w/v) bisacrylamide stock (e.g., Bio‐Rad)
  • 10% (w/v) ammonium persulfate (APS; e.g., Bio‐Rad): 100 mg in 1 ml water, prepared just before gel casting
  • 1.5 M Tris⋅Cl, pH 8.8 (see recipe)
  • 10% (w/v) sodium dodecyl sulfate (SDS; see recipe)
  • TEMED (N,N,N′,N′‐tetramethylethylenediamine; e.g., Bio‐Rad)
  • Isobutanol, water saturated
  • 1.0 M Tris⋅Cl, pH 6.8 (see recipe)
  • Mini‐Protean II Electrophoresis Cell (Bio‐Rad)
  • Casting stand
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (see appendix 3A)
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Literature Cited

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