Measurement of Apoptosis and Other Forms of Cell Death

Jagan Muppidi1, Melissa Porter1, Richard M. Siegel1

1 National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.17
DOI:  10.1002/0471142735.im0317s59
Online Posting Date:  May, 2004
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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. This unit presents a set of assays for these purposes, many of which are technically very simple and ideally suited to the study of hematopoietic cells. The first basic protocol allows the qualitative and quantitative assessment of apoptosis in lymphocyte cell cultures using light or fluorescent microscopy. Three protocols follow that are designed to detect nuclear DNA fragmentation and support protocols describe methods to radiolabel the DNA and cytoplasm of the cells to be tested. Techniques that quantitate apoptotic cells using flow cytometry are then described and support protocols provide methods for priming T cell clones and freshly isolated lymph node cells, respectively, for T cell receptor (TCR)‐induced apoptosis. Quantitative detection of DNA fragmentation in apoptotic cells is also described. TdT‐mediated dUTP‐biotin nick end‐labeling (TUNEL) methods are provided for the detection of apoptotic cells, along with procedures for the flow cytometric quantitation of apoptotic cells using TUNEL, and TUNEL, staining of tissue sections to identify apoptotic cells. Since much remains incompletely understood about the molecular pathways of programmed death, and it is probably best to perform more than one of the basic protocols to confirm an observation of apoptotic cell death.

     
 
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Table of Contents

  • Section IV: Related Isolation Procedures and Functional Assays
  • Basic Protocol 1: Microscopic Quantitation of Cell Viability Using Vital and Fluorescent Dyes
  • Basic Protocol 2: Flow Cytometric Quantitation of Cell Death
  • Alternate Protocol 1: Annexin V Staining for Measurement of Phosphotidylserine Exposure
  • Alternate Protocol 2: Flow Cytometric Assessment of DNA Fragmentation with the TUNEL Assay
  • Alternate Protocol 3: Flow Cytometric Assessment of Caspase Activation
  • Alternate Protocol 4: Determination of Apoptosis Using the Sub‐G0/G1 DNA Peak
  • Support Protocol 1: Priming Mouse or Human T Cells for Apoptosis Assays
  • Basic Protocol 3: Quantitation of DNA Fragmentation and Lysis
  • Support Protocol 2: Nuclear Radiolabeling with [125I]UdR and 51Cr
  • Support Protocol 3: Nuclear Radiolabeling with [3H]TdR
  • Alternate Protocol 5: Quantitation of DNA Fragmentation in Cells Containing Radiolabeled DNA
  • Alternate Protocol 6: Qualitative Analysis of Internucleosomal DNA Fragmentation by Agarose Gel Electrophoresis
  • Basic Protocol 4: Assessment of the Pro‐ or Anti‐Apoptotic Properties of a cDNA Clone
  • Basic Protocol 5: Analysis of Death Receptor Signaling Complexes
  • Basic Protocol 6: In Situ Detection of Apoptotic Cells in Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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

  Materials
  • Dye mix (see recipe)
  • Cells suspension in PBS ( appendix 2A)
  • 96‐well round‐bottom plate
  • Microscope slide
  • No. 1–thickness coverslip or coverslip precoated with poly‐L‐lysine (for nonadherent cell; Sigma)
  • Epifluorescent microscope (unit 21.2) equipped with 40× or 63× objective and appropriate filter sets (Table 3.17.2)
    Table 3.7.2   MaterialsFilter Sets for Fluorescence Microscopy

    Excitation filter (nm) Emission filter (nm)
    Narrow‐band FITC 450 to 490 520 to 560
    Rhodamine 510 to 560 590 to 700
    Wide band FITC 450 to 490 520 to 700

CAUTION: Acridine orange and ethidium bromide have been found by the Ames test to be highly mutagenic and should be handled with care.

Basic Protocol 2: Flow Cytometric Quantitation of Cell Death

  Materials
  • Cells in tissue culture dish or suspension
  • 100 µg/ml propidium iodide (PI) in PBS (store protected from light up to 6 months at room temperature; see appendix 2A for PBS)
  • FACS buffer (see recipe)
  • Additional reagents and equipment for flow cytometric analysis and analysis of flow cytometric data (unit 5.4)

Alternate Protocol 1: Annexin V Staining for Measurement of Phosphotidylserine Exposure

  • Apoptotic stimulus (e.g., anti‐TCR mAb, anti‐Fas Ab)
  • Annexin V labeling buffer (see recipe)
  • Fluorophore‐conjugated annexin V: annexin V conjugated to FITC, PE, or equivalent (e.g., PharMingen, Molecular Probes)
  • Paraformaldehyde fixing solution (see recipe)
  • 24‐ or 96‐well flat‐bottomed plates
  • 12 × 75–mm round‐bottom polystyrene tubes, 6‐ml capacity
  • Sorvall RT6000B centrifuge and H1000B rotor (or equivalent)

Alternate Protocol 2: Flow Cytometric Assessment of DNA Fragmentation with the TUNEL Assay

  Materials
  • Cells for analysis
  • PBS ( appendix 2A)
  • 95% ethanol, ice cold
  • Paraformaldehyde fixation solution (see recipe)
  • TdT reaction buffer (see recipe)
  • TdT/biotin‐dUTP mix (see recipe)
  • Fluorescein isothiocyanate (FITC)–conjugated streptavidin (Jackson Immunoresearch)
  • 12 × 75–mm round‐bottom centrifuge tubes
  • Sorvall Legend RT centrifuge (or equivalent) with swinging‐bucket rotor
  • Additional reagents and equipment for immunofluorescence staining (optional; unit 5.3) and flow cytometric analysis (unit 5.4)

Alternate Protocol 3: Flow Cytometric Assessment of Caspase Activation

  • Paraformaldehyde fixation solution (see recipe)
  • Permeablization/wash buffer (see recipe)
  • Anti‐active caspase‐3 antibody (clone C92‐605; BD PharMingen)
  • PBS ( appendix 2A)

Alternate Protocol 4: Determination of Apoptosis Using the Sub‐G0/G1 DNA Peak

  Materials
  • Cells
  • FACS buffer (see recipe), 4°C
  • 70% or 100% ethanol, 4°C
  • FACS buffer containing RNase A and PI (see recipe)
  • 1 mg/ml RNase A (use DNase free RNase or boil the RNase solution for 5 min and let slowly cool to room temperature before storing frozen in aliquots at −20°C)
  • 1 mg/ml propidium iodide (PI) in PBS ( appendix 2A)

Support Protocol 1: Priming Mouse or Human T Cells for Apoptosis Assays

  Materials
  • Single‐cell suspension of lymph node or human peripheral blood mononuclear cells (unit 3.1)
  • Complete RPMI medium ( appendix 2A)
  • 1 mg/ml concanavalin A (Con A; store up to 6 months at 4°C and pass through a 0.2‐µm filter before use)
  • 200 mg/ml α‐methyl‐D‐mannoside in complete RPMI medium or PBS ( appendix 2A): store up to 6 months at 4°C and pass through a 0.2‐µm filter before use
  • Anti‐CD3—i.e., clone 145‐2c11 (mouse) or HIT2a (human); BD Pharmingen—in PBS
  • PBS ( appendix 2A)
  • RMPI‐10 ( appendix 2A) containing 100 U/ml IL‐2 (R & D Systems)
  • Anti‐Fas clone Jo‐2 biotin (BD Pharmingen)
  • Streptavidin
  • APO‐1‐3 (Kamiya Biochemicals)
  • Soluble protein A
  • 15‐ml conical tube
  • Sorvall Legend RT centrifuge with H‐1000B swinging bucket rotor (or equivalent)
  • 6‐well tissue culture dish or tissue culture flask
  • 24‐ or 96‐well plate
  • Additional reagents and equipment for counting cells ( appendix 3A) and removal of dead cells using a one‐step gradient (unit 3.1)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Quantitation of DNA Fragmentation and Lysis

  Materials
  • T cells with nuclear ([125I]UdR) and cytoplasmic (51Cr) radiolabeling (see protocol 9) in complete RPMI medium
  • Complete RPMI medium ( appendix 2A), room temperature and ice‐cold
  • Unlabeled cytotoxic T lymphocytes (CTL; unit 3.11; optional)
  • TTE solution: TE buffer, pH 7.4 ( appendix 2A) containing 0.2% Triton X‐100 (store up to 6 months at 4°C)
  • Sorvall RT6000 or equivalent tabletop centrifuge
  • Additional reagents and equipment for killing target cells with CTL (unit 3.11)

Support Protocol 2: Nuclear Radiolabeling with [125I]UdR and 51Cr

  Materials
  • Cells suspensions
  • Complete RPMI‐10 medium ( appendix 2A)
  • 1 mCi/ml iododeoxyuridine ([125I]UdR) in aqueous solution (∼2000 Ci/mmol; ICN Biomedicals)
  • 5 mCi/ml 51Cr, as Na 2CrO 4 in saline (200 to 900 Ci/g; ICN Biomedicals; optional)
  • RPMI 1640 medium (Life Technologies), 37°C
  • 17 × 100–mm polystyrene tissue culture tubes (Falcon)
  • IEC model CRU‐5000 centrifuge (or equivalent)
  • γ‐Scintillation counter

Support Protocol 3: Nuclear Radiolabeling with [3H]TdR

  Materials
  • Cells to be labeled
  • Complete RPMI‐10 medium ( appendix 2A)
  • 1 mCi/ml [methyl‐3H]thymidine ([3H]TdR) in aqueous solution (2.0 Ci/mmol; ICN Biomedicals)
  • RPMI 1640 medium (Life Technologies), 37°C

Alternate Protocol 5: Quantitation of DNA Fragmentation in Cells Containing Radiolabeled DNA

  • [125I]UdR‐ or [3H]TdR‐labeled cells/ml (see protocol 9Support Protocols 2 or protocol 103) in complete RPMI‐10 medium ( appendix 2A)
  • Scintillation cocktail compatible with aqueous mixtures (for [3H]TdR label)
  • γ‐Scintillation counter (for [3H]TdR label)

Alternate Protocol 6: Qualitative Analysis of Internucleosomal DNA Fragmentation by Agarose Gel Electrophoresis

  • 5 M NaCl, 4°C
  • 2‐propanol, 4°C
  • 70% ethanol, 4°C
  • TE buffer, pH 7.4 and 8.0 ( appendix 2A)
  • 10× loading buffer (unit 10.4)
  • TBE electrophoresis buffer (unit 10.4)
  • Cotton‐tipped swab
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4) and autoradiography ( appendix 3J; optional)

Basic Protocol 4: Assessment of the Pro‐ or Anti‐Apoptotic Properties of a cDNA Clone

  Materials
  • Jurkat or other cell suspension in complete media
  • Complete medium
  • cDNA
  • Apoptotic stimulus (i.e., anti‐Fas or staurosporine)
  • Anti‐human Fas/protein A master mix (see recipe)
  • zVAD‐fmk caspase inhibitor (Enzyme Systems Products)
  • 6‐well plate or small flask
  • 24‐well plates
  • 0.4‐cm gap cuvette (Bio‐Rad)
  • BTX electroporator (http://www.btxonline.com) or equivalent
  • Additional reagents and equipment for FACS analysis using annexin V–conjugated dye or fluorescent‐caspase substrate (see protocol 3)

Basic Protocol 5: Analysis of Death Receptor Signaling Complexes

  Materials
  • Cell suspension
  • PBS ( appendix 2A), room temperature and 4°C
  • Anti‐human Fas (Apo1‐3, Kamiya Biochemicals) or recombinant human FasL (rhFasL, Alexis)
  • Lysis buffer (see recipe), room temperature and 4°C
  • Protein G agarose (Roche)
  • Antibodies for immunoblotting:
    • Anti‐caspase‐8 clone C15 (Alexis 804‐429)
    • Anti‐FADD (BD Pharmingen)
    • Anti‐Fas (Santa Cruz, C20)
  • Appropriate secondary antibody
  • End‐over‐end rotator
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and immunoblotting (unit 8.10)

Basic Protocol 6: In Situ Detection of Apoptotic Cells in Tissue Sections

  Materials
  • Fresh tissue for analysis
  • 1% (w/v) paraformaldehyde in PBS (dissolve by stirring with low heat overnight and filter before use)
  • Acetone
  • Tris‐buffered saline (TBS; appendix 2A)
  • 0.1% or 0.01% (v/v) H 2O 2 in TBS
  • TdT reaction buffer (see recipe)
  • TdT/digoxigenin‐dUTP mix (see recipe)
  • 2% (v/v) horse serum or FBS in TBS
  • Sheep anti‐digoxigenin primary antibody solution (see recipe)
  • HRPO‐conjugated anti‐sheep secondary antibody solution (see recipe)
  • AEC substrate working solution (see recipe)
  • Mayer's hematoxylin (Sigma)
  • Crystal Mount (Fisher)
  • Hydrophobic‐barrier slide marker (e.g., PAP Pen; Research Products International)
  • Coplin jars or staining trays
  • Humidified container (see recipe)
  • Additional reagents and equipment for preparing frozen sections (unit 21.4)
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Figures

  •   FigureFigure 3.17.1 Examples of apoptotic and necrotic cellular morphology. Giemsa‐stained samples of Jurkat T lymphoma cells were left either (A) untreated, (B) infected with HIV‐1, which induces primarily necrotic cell morphology with disrupted nucleus and cytoplasm, or (C) treated with anti‐Fas mAb, which induces nuclear condensation typical of apoptosis. Hoechst‐stained samples of thymocytes treated with either (D) vehicle, (E) 46°C heat shock, which induces primarily necrosis as measured by annexin and PI staining, or (F) 100 ng/ml dexamethasone for 4 hr, which induces apoptosis (Table ). Cells were stained with 1 µg/ml Hoechst 33342 and photographed with an epifluorescence microscope at 630× magnification. Note the many condensed and featureless nuclei in panel F. Nuclear changes of (E) heat‐shocked thymocytes undergo relatively little morphological change.
  •   FigureFigure 3.17.2 Annexin V and PI staining of apoptotic cells. Jurkat cells were incubated for 16 hr at 1 × 106 cells/ml in the presence or absence of 100 ng/ml rhFasL (Alexis Biochemicals) and 1 µg/ml anti‐FLAG antibody to cross‐link the stimulus. Cells were stained with annexin V FITC and propidium iodide (PI) and analyzed by FACS.
  •   FigureFigure 3.17.3 Detection of apoptotic cells by the TUNEL method using flow cytometric analysis. Thymocytes from 6‐week‐old C57BL/6 mice were stained for apoptotic cells with FITC by the TUNEL method before and after an 18‐hr incubation in RPMI‐10 at 37°C. (A) Dot plot depicting forward‐ (FSC) versus side‐scatter (SSC) profile of cells after incubation. The bulk of thymocytes (in the gate R1) are easily distinguished from smaller cellular debris (low FSC) and larger cell clumps (high FSC). (B) Dot plot depicting profile of FITC fluorescence versus FSC for cells after incubation. Most of the TUNEL‐stained apoptotic cells are slightly smaller than unstained live cells. (C) Histogram depicting cells before incubation in the gate R1. Very few apoptotic cells are detected. (D) Histogram depicting cells after incubation in the gate R1. Apoptotic cells (brightly stained by TUNEL) are detected.
  •   FigureFigure 3.17.4 Example of protection from apoptosis conferred by a transfected gene. Jurkat cells were transiently transfected with plasmids encoding GFP or a vFLIP‐GFP fusion protein. vFLIP is a known inhibitor of Fas‐induced apoptosis. After overnight incubation, one group of cells were treated with anti‐Fas mAb Apo‐1‐3 while another was not. The groups were then analyzed by flow cytometry for GFP and annexin V‐PE as shown. Note the relative absence of annexin V‐positive GFP positive cells in the bottom right plot from cells transfected with vFLIP and treated with anti‐Fas mAb.
  •   FigureFigure 3.17.5 In situ detection of apoptotic cells in thymus sections by the TUNEL technique. Sections were counterstained with hematoxylin and photomicrographed at 200× magnification (also see Anticipated Results). (A) Thymus from 5‐week‐old I‐E+/Vβ5 transgenic mouse. Small apoptotic cells are scattered in the cortex (cor) but are unusually prominent in the medulla (med) as large aggregates. (B) Thymus from 5‐week‐old I‐E‐/Vβ5 transgenic mouse. This is similar to the normal mouse thymus; apoptotic cells are apparent in the cortex but rare in the medulla.
  •   FigureFigure 3.17.6 Schematic of a humidified (moist) chamber.

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Literature Cited

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