Flow Cytometry of Apoptosis

Piotr Pozarowski1, Jerzy Grabarek2, Zbigniew Darzynkiewicz3

1 School of Medicine, Lublin, null, 2 Pomeranian School of Medicine, Szczecin, Poland, 3 New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.19
DOI:  10.1002/0471142956.cy0719s25
Online Posting Date:  August, 2003
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Abstract

Application of flow cytometry to the study of cell death has three goals: identification and quantification of dead and dying cells; discrimination between apoptotic and necrotic modes of cell death; and elucidation of mechanisms involved in cell death. This massively detailed unit by a pioneer in the field brings together the most common flow cytometric methods for the study of apoptosis, covering a wide variety of apoptotic indices, from loss of membrane potential, caspase activation, and phosphatidyl exposure to DNA fragmentation and tissue transglutaminase activation. The authors also present their recently developed protocol, analogous to the FLICA approach for caspases, for the detection of serine proteases (‘serpases’). The protocols are accompanied by extensive commentary discussion of applicability, strategic planning, problems, and pitfalls, plus a comprehensive list of references.

Keywords: flow cytometry; apoptosis; membrane potential; caspase activation; annexin V; serine proteases; DNA fragmentation; FLICA; TUNEL

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Mitochondrial Transmembrane Potential (Δψm) Measured by Rhodamine 123 or DiOC6(3) Fluorescence
  • Basic Protocol 2: Immunocytochemical Detection of Activated Caspases by Zenon Technology
  • Basic Protocol 3: Detection of Apoptotic Cells Using Fluorochrome‐Labeled Inhibitors of Caspases (FLICAs)
  • Basic Protocol 4: Determination of Poly(ADP‐Ribose) Polymerase (PARP) Cleavage
  • Basic Protocol 5: Annexin V Binding
  • Basic Protocol 6: DNA Fragmentation: Detection of Cells with Fractional (Sub‐G1) DNA Content Using PI
  • Alternate Protocol 1: DNA Fragmentation: Detection of Cells with Fractional (“Sub‐G1”) DNA Content Using DAPI
  • Basic Protocol 7: DNA Fragmentation: Detection of DNA Strand Breaks (TUNEL Assay)
  • Basic Protocol 8: Detection of Tissue Transglutaminase Activation by Cell Resistance to Detergents
  • Alternate Protocol 2: Detection of TGase 2 Activation by Fluoresceinated Cadaverine (F‐CDV) Binding
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Mitochondrial Transmembrane Potential (Δψm) Measured by Rhodamine 123 or DiOC6(3) Fluorescence

  Materials
  • Cells of interest in appropriate complete culture medium
  • 10 µM rhodamine 123 (R123; see recipe) or 10 µM DiOC 6(3) (see recipe for 0.1 mM stock solution) or 0.2 mM JC‐1 stock solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1 mg/ml propidium iodide (PI; Molecular Probes) in distilled water; store at 4°C in the dark
  • 12 × 75–mm tubes suitable for flow cytometer
  • Flow cytometer with 488‐nm excitation and filters for collection of green, orange, and red fluorescence

Basic Protocol 2: Immunocytochemical Detection of Activated Caspases by Zenon Technology

  Materials
  • Cells of interest (see appendix 3B for culture techniques), both untreated (control) and induced to apoptosis (e.g., exponentially growing HL‐60 cells incubated 2 to 4 hr with 0.15 µM camptothecin)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fixatives:
    • 1% (v/v) methanol‐free formaldehyde (Polysciences) in PBS, 0° to 5°C
    • 4% (v/v) methanol‐free formaldehyde (Polysciences) in PBS, room temperature
    • 70% (v/v) ethanol, –20°C
  • Rinse solution (see recipe)
  • Primary antibody: cleaved (activated) caspase‐3 antibody (Cell Signaling Technology, cat. no. 9661)
  • Zenon Alexa Fluor 488 rabbit IgG labeling kit (Molecular Probes, cat. no. Z‐25302)
  • 10% (v/v) Triton X‐100 in PBS
  • DNA staining solution with PI (see recipe)
  • 12 × 75–ml tubes suitable for use on flow cytometer
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 3: Detection of Apoptotic Cells Using Fluorochrome‐Labeled Inhibitors of Caspases (FLICAs)

  Materials
  • Cells of interest (see appendix 3B for culture techniques)
  • Medium supplemented with 10% (v/v) serum or 1% (w/v) serum albumin
  • FLICA kit (Immunochemistry Technologies) containing:
    • FAM‐VAD‐FMK reagent (see recipe)
    • Fixative
    • Hoechst stain
  • Rinse solution: 1% (w/v) BSA in PBS ( appendix 2A)
  • 1 mg/ml propidium iodide (PI; Molecular Probes) in distilled water; store at 4°C in the dark
  • 12 × 75–ml tubes suitable for use on flow cytometer
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 4: Determination of Poly(ADP‐Ribose) Polymerase (PARP) Cleavage

  Materials
  • Cells of interest
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% methanol‐free formaldehyde (Polysciences) in PBS ( appendix 2A)
  • 70% ethanol
  • 0.25% (v/v) Triton X‐100 (Sigma) in PBS ( appendix 2A); store at 4°C
  • PBS/BSA solution: 1% (w/v) bovine serum albumin (Sigma) in PBS; store at 4°C
  • Anti‐PARP p85 antibody (Promega anti‐PARP‐85 fragment, rabbit polyclonal)
  • Fluorescein‐conjugated anti‐rabbit immunoglobulin antibody (Dako)
  • 1 mg/ml propidium iodide (PI; Molecular Probes) in distilled water; store at 4°C in the dark
  • RNase solution ( appendix 2A)
  • 12 × 75–mm centrifuge tubes suitable for use on the flow cytometer
  • Pasteur pipets
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 5: Annexin V Binding

  Materials
  • Cells of interest
  • Fluorescein‐conjugated annexin V (see recipe) in binding buffer (see recipe)
  • 1 mg/ml propidium iodide (PI; Molecular Probes) in distilled water; store at 4°C in the dark
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 6: DNA Fragmentation: Detection of Cells with Fractional (Sub‐G1) DNA Content Using PI

  Materials
  • Cells of interest
  • PBS ( appendix 2A)
  • 70% ethanol
  • DNA extraction buffer (see recipe)
  • DNA staining solution with PI (see recipe)
  • 12 × 75–mm tubes suitable for use on the flow cytometer
  • Flow cytometer with 488‐nm excitation and filter for collection of red fluorescence

Alternate Protocol 1: DNA Fragmentation: Detection of Cells with Fractional (“Sub‐G1”) DNA Content Using DAPI

  • DNA staining solution with DAPI (see recipe)
  • Flow cytometer equipped with UV excitation and filter for collection of blue fluorescence

Basic Protocol 7: DNA Fragmentation: Detection of DNA Strand Breaks (TUNEL Assay)

  Materials
  • Cells of interest
  • 1% (v/v) methanol‐free formaldehyde (Polysciences) in PBS ( appendix 2A), pH 7.4 (primary fixative)
  • PBS ( appendix 2A)
  • 70% ethanol (secondary fixative)
  • 5× TdT reaction buffer (see recipe)
  • 2 mM BrdUTP (Sigma) in 50 mM Tris·Cl, pH 7.5
  • TdT in storage buffer (both from Roche Diagnostics), 25 U in 1 µl
  • 10 mM cobalt chloride (CoCl 2; Roche Diagnostics)
  • Rinsing buffer: PBS with 0.1% (v/v) Triton X‐100 and 0.5% (w/v) BSA
  • FITC‐conjugated anti‐BrdU MAb in PBS ( appendix 2A; see recipe)
  • PI staining buffer: PBS with 5 µg/ml PI and 200 µg/ml DNase‐free RNase
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence

Basic Protocol 8: Detection of Tissue Transglutaminase Activation by Cell Resistance to Detergents

  Materials
  • Cells of interest
  • DAPI/sulforhodamine 101/detergent solution (see recipe)
  • Flow cytometer equipped with UV excitation and filters for collection of blue and red fluorescence

Alternate Protocol 2: Detection of TGase 2 Activation by Fluoresceinated Cadaverine (F‐CDV) Binding

  Materials
  • Fluoresceinated cadaverine solution (F‐CDV; see recipe)
  • Cells of interest
  • 100% methanol
  • DNA staining solution with PI (see recipe)
  • Flow cytometer with 488‐nm excitation and filters for collection of green and red fluorescence
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Figures

  •   FigureFigure 7.19.1 Changes in light scattering properties of cells undergoing apoptosis. HL‐60 cells were untreated (left panel) or treated 3 hr with TNF‐α and cycloheximide (CHX) to induce apoptosis (right panel). Cell population A in the treated culture (right panel) represents cells that have light scattering properties similar to those of untreated cells. Early apoptotic cells (B) have diminished forward scatter and are very heterogenous with respect to side scatter. Late apoptotic cells (C) have both forward and side scatter diminished.
  •   FigureFigure 7.19.2 Detection of caspase activation during apoptosis by affinity labeling of their active center with FAM‐VAD‐FMK combined with supravital staining with PI. Apoptosis was induced in cultures by administering TNF‐α+CHX or CPT as described (Smolewski et al., 2002). FAM‐VAD‐FMK (20 µM) was included in cultures from the onset of treatment (TNF+FLICA; CPT+FLICA). The TNF‐α‐ and CPT‐treated cultures were maintained 24 and 48 hr, respectively. Cells from all cultures were supravitally stained with 1 µg/ml PI 5 min prior to fluorescence measurement. Green (FAM‐VAD‐FMK) and red (PI) cellular fluorescence was measured by flow cytometry. Four cell subpopulations (AD) can be identified, differing in their capability to bind FAM‐VAD‐FMK and PI (see text). They represent the progressive stages of apoptosis, starting with caspase activation (B), loss of plasma‐membrane ability to exclude PI (C), and loss of caspase reactivity with FAM‐VAD‐FMK (D).
  •   FigureFigure 7.19.3 Detection of the collapse of mitochondrial electrochemical potential (ϕm) by rhodamine 123 (R123). HL‐60 cells were untreated (control; left panel) or treated 3 hr with TNF‐α and CHX (right panel) to induce apoptosis. The cells were then incubated with R123 and PI according to . The early apoptotic cells have diminished green fluorescence of R123 but exclude PI (cell population B). The late apoptotic (also necrotic) cells are stained strongly by PI (population C).
  •   FigureFigure 7.19.4 Immunocytochemical detection of caspase‐3 activation using antibody reactive with the activated (cleaved) caspase‐3. Apoptosis of HL‐60 cells was induced by topotecan (TPT), an analog of CPT. Zenon technology (Haugland, ) was used to detect caspase‐3 as described in . Top and bottom insets in each panel show cellular DNA content frequency histograms of cells with activated and nonactivated caspase‐3, respectively. Note that S‐phase cells preferentially contain activated caspase‐3 after induction of apoptosis by TPT.
  •   FigureFigure 7.19.5 Binding of fluorochrome‐labeled inhibitor of caspases (FLICA; FAM‐VAD‐FMK) and PI during apoptosis. Apoptosis of HL‐60 cells was induced by TPT. The cells were stained according to . Green (FAM‐VAD‐FMK) and red (PI) cellular fluorescence was measured by flow cytometry. Four cell subpopulations (A to D) can be identified, differing in their capability to bind FAM‐VAD‐FMK and PI. They represent sequential stages of apoptosis, starting with binding of FAM‐VAD‐FMK (B), loss of plasma membrane integrity to exclude PI (C), and loss of reactivity with FAM‐VAD‐FMK (D).
  •   FigureFigure 7.19.6 Identification of apoptotic cells by flow cytometry based on the immunocytochemical detection of the 85‐kDa PARP cleavage fragment. To induce apoptosis, HL‐60 cells were treated 60 min with TNF‐α in the presence of CHX (Li and Darzynkiewicz, ). PARPp85 was detected immunocytochemically and DNA was counterstained with PI, as described in .
  •   FigureFigure 7.19.7 Detection of early and late apoptotic cells after staining with annexin V–FITC and PI. To induce apoptosis, HL‐60 cells were treated 2 hr with TNF‐α and CHX. Untreated (control; left panel) and TNF‐α‐treated (right panel) cells were then stained with annexin V‐FITC and PI.
  •   FigureFigure 7.19.8 Detection of apoptotic cells by flow cytometry based on cellular DNA content analysis. (A) Normal cell plot. (B) To induce apoptosis, HL‐60 cells were treated with the DNA topoisomerase II inhibitor fostriecin (Hotz et al., ). Cells were fixed in 70% ethanol, suspended in high‐molarity phosphate buffer to extract fragmented DNA, and then stained with PI. A subpopulation of apoptotic cells (Ap) with fractional (sub‐diploid) DNA content, i.e., with DNA index (DI) <1.0 (sub‐G1 cells), is apparent. Note also the increase in the proportion of S‐phase cells in the nonapoptotic population. (C) The fragmented DNA extracted from the apoptotic cells by the buffer was subjected to gel electrophoresis (Gong et al., ). Note “laddering” that reflects preferential DNA cleavage at internucleosomal sections, the characteristic feature of apoptosis (Arends et al., ).
  •   FigureFigure 7.19.9 Detection of apoptotic cells by flow cytometry based on the presence of DNA strand breaks. To induce apoptosis, HL‐60 cells were treated 120 or 150 min with CPT (Li and Darzynkiewicz, ). DNA strand breaks were labeled with BrdUTP using exogenous terminal deoxynucleotidyl transferase. The cell cycle distribution of both apoptotic and nonapoptotic cell subpopulations can be estimated based on the DNA content of individual cells. Note that in analogy to PARP cleavage (Fig. ), preferentially S‐phase cells undergo apoptosis following CPT treatment.
  •   FigureFigure 7.19.10 Detection of tissue transglutaminase (TGase 2) activation during apoptosis by the acquired resistance of the cytoplasmic proteins to detergent. Bivariate distributions illustrating red fluorescence of sulforhodamine 101 (protein content) versus blue fluorescence of DAPI (DNA content) of HL‐60 cells, untreated (A) or exposed to hyperthermia (72 hr at 41.5°C) in the absence (B) and presence (C) of the cytotoxic RNase onconase (1.67 µM; Grabarek et al., ). Following cell lysis by Triton X‐100 and staining with DAPI and sulforhodamine 101, the isolated nuclei of nonapoptotic cells from control culture (A) show low and uniform intensity of red fluorescence, reflecting low protein content. Subpopulations of apoptotic cells in B and C have their cytoplasmic protein crosslinked and therefore are resistant to detergent. They stain intensely with sulforhodamine 101. Note differences in DNA content (cell cycle) distribution of the cells with activated (top insets; cells gated above the dashed line) versus nonactivated TGase 2 (bottom insets; cells gated below the dashed line) in B and C. Percentage of cells with activated and non‐activated TGase 2 in the respective cultures is indicated in each panel.
  •   FigureFigure 7.19.11 Detection of TGase 2 activity in HL‐60 cells using FITC‐conjugated cadaverine (F‐CDV) as the enzyme substrate. Cultures of untreated (A) and hyperthermia (5 hr at 41.5°C)‐treated (B) HL‐60 cells were incubated 5 hr with 100 µM F‐CDV. Cells were then fixed and their DNA was counterstained with PI in the presence of RNase. Note the presence in the untreated culture (A) of few cells that incorporated F‐CDV (spontaneous apoptosis), and large numbers of F‐CDV‐labeled cells in the treated culture (B). Note also that some apoptotic cells with fractional DNA content (“sub‐G1” subpopulation) in the treated culture do not show incorporation of F‐CDV (arrow). Percentage of cells with activated and nonactivated TGase 2 in the respective cultures is indicated. The inset shows the cellular DNA content distribution histogram of all cells (Grabarek et al., ).

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