Measurement and Characterization of Apoptosis by Flow Cytometry

William Telford1, Karen Tamul2, Jolene Bradford3

1 Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 2 EMD Millipore, Inc., Billerica, Massachusetts, 3 Thermo Fisher Scientific, Inc., Eugene, Oregon
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.49
DOI:  10.1002/cpcy.1
Online Posting Date:  July, 2016
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Abstract

Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down‐regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti‐cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal‐transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation. These assays are particularly powerful when combined into multicolor assays determining several apoptotic characteristics simultaneously. The multiparametric nature of flow cytometry makes this technology particularly suited to measuring apoptosis. In this unit, we will describe the four main techniques for analyzing caspase activity in apoptotic cells, combined with annexin V and cell permeability analysis. These relatively simple multiparametric assays are powerful techniques for assessing cell death. © 2016 by John Wiley & Sons, Inc.

Keywords: 7‐aminoactinomycin D; annexin V; apoptosis; caspase; covalent viability probe; fluorogenic caspase substrate; propidium iodide

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: FLICA Caspase Substrates with Annexin V And DNA Dye
  • Alternate Protocol 1: FLICA Caspase Substrates with Covalent Viability Probe
  • Alternate Protocol 2: FLICA Caspase Substrates with Annexin V and Covalent Viability Probe
  • Basic Protocol 2: PhiPhiLux Caspase Substrates with Annexin V and DNA Dye
  • Alternate Protocol 3: PhiPhiLux Caspase Substrates with Annexin V and Covalent Viability Probe
  • Basic Protocol 3: CellEvent Green or NucView 488 Substrates with Annexin V and DNA Dye
  • Alternate Protocol 4: CellEvent Green or NucView 488 Substrates with Annexin V and Covalent Viability Probe
  • Basic Protocol 4: Immunolabeling for Active Caspase 3
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: FLICA Caspase Substrates with Annexin V And DNA Dye

  Materials
  • Propidium iodide, 7‐aminoactinomycin D (7‐AAD; see recipe), or other DNA dye (see Table 9.49.1 and annotation to step 1, below)
  • Wash buffer (see recipe)
  • Cells to be analyzed
  • 30× FLICA fluorogenic caspase 3/7 substrate (see recipe)
  • 100 μg/ml Alexa Fluor 647 or Pacific Blue–conjugated annexin V (see recipe)
  • Sample tubes, typically 12 × 75–mm tubes compatible with flow cytometry
  • Centrifuge capable of 400 × g centrifugation
  • Flow cytometer
Table 9.9.1   MaterialsDNA Binding Dyes Used for Cell Permeability Assessment by Flow Cytometry, with Excitation and Emission Information

DNA dye Excitation max (λ) Laser line (nm) Emission max (λ), nm
Propidium iodide a
7‐aminoactinomycin D a 546 488 nm, 532 nm, 552 nm, 561 nm 647
SYOTX AADvanced b 535 488 nm, 532 nm, 552 nm, 561 nm 617
YO‐PRO‐3 b 491 488 nm 509
SYTOX Green b 504 488 nm 523
DRAQ7 c 600, 646 633, 640 nm 697
TO‐PRO‐3 b 642 633 nm, 640 nm 661
SYTOX Red c 635 633 nm, 640 nm 655
PO‐PRO‐1 b 435 405 nm 455
DAPI a 358 355 nm, 375 nm, 405 nm 461
Hoechst 33258 a 352 355 nm, 375 nm, 405 nm 461
SYTOX Blue b 445 405 nm 470

 aSeveral manufacturers.
 bThermo Fisher Scientific, Inc. (all names are trademarked).
 cBiostatus Ltd. (name is trademarked).

Alternate Protocol 1: FLICA Caspase Substrates with Covalent Viability Probe

  Additional Materials (also see protocol 1)
  • Covalent viability probe (lyophilized) with appropriate fluorochrome (see annotation to step 1, below)
  • Dimethylsulfoxide (DMSO)
  • PBS or HBSS with calcium/magnesium
  • 2% (w/v) paraformaldehyde in PBS with Ca and Mg

Alternate Protocol 2: FLICA Caspase Substrates with Annexin V and Covalent Viability Probe

  Additional Materials (also see protocol 1)
  • Covalent viability probe with appropriate fluorochrome (see protocol 2, step 1 annotation)
  • PBS or HBSS with calcium/magnesium, serum‐free

Basic Protocol 2: PhiPhiLux Caspase Substrates with Annexin V and DNA Dye

  Materials
  • Propidium iodide, 7‐aminoactinomycin D (7‐AAD; see recipe), or other DNA dye (also see Table 9.49.1 and protocol 1, step 1 annotation)
  • Wash buffer (see recipe)
  • Cells to be analyzed
  • 10 μM PhiPhiLux G1D2 caspase substrate (see recipe)
  • 100 μg/ml Alexa Fluor 647– or Pacific Blue–conjugated annexin V (see recipe)
  • Sample tubes, typically 12 × 75–mm tubes compatible with flow cytometry
  • Centrifuge capable of 400 × g centrifugation
  • Flow cytometer

Alternate Protocol 3: PhiPhiLux Caspase Substrates with Annexin V and Covalent Viability Probe

  Additional Materials (also see Basic Protocols protocol 11 and protocol 42)
  • Covalent viability probe with appropriate fluorochrome (see protocol 2, step 1 annotation)
  • PBS or HBSS with calcium/magnesium, serum‐free

Basic Protocol 3: CellEvent Green or NucView 488 Substrates with Annexin V and DNA Dye

  Materials
  • Propidium iodide, 7‐aminoactinomycin D (7‐AAD; see recipe), or other DNA dye (see protocol 1, step 1 annotation)
  • Wash buffer (see recipe)
  • Cells to be analyzed
  • 500 μM CellEvent Green or NucView 488 substrate (see recipe)
  • 100 μg/ml Alexa Fluor 647– or Pacific Blue–conjugated annexin V (see recipe)
  • Sample tubes, typically 12 × 75–mm tubes compatible with flow cytometry
  • Centrifuge capable of 400 × g centrifugation
  • Flow cytometer

Alternate Protocol 4: CellEvent Green or NucView 488 Substrates with Annexin V and Covalent Viability Probe

  Additional Materials (also see Basic Protocols protocol 11 to protocol 63)
  • Covalent viability probe with appropriate fluorochrome (see protocol 2, step 1 annotation)
  • PBS or HBSS with calcium/magnesium, serum‐free

Basic Protocol 4: Immunolabeling for Active Caspase 3

  Materials
  • Cells to be analyzed
  • PBS or HBSS, serum‐free
  • Covalent viability probe with appropriate fluorochrome (see protocol 2, step 1 annotation)
  • Fixative and permeabilization reagents (see recipe):
  • Permeabilization buffer [PBS with 2% (v/v) fetal bovine serum and 0.1% (v/v) Tween 20)
  • or
  • Commercial fixation/permeabilization buffer system (i.e., BD Biosciences Pharmingen Fix/Perm and Perm/Wash system)
  • 2% paraformaldehyde in PBS
  • Anti‐caspase monoclonal antibody (see recipe)
  • Sample tubes, typically 12 × 75–mm tubes compatible with flow cytometry
  • Centrifuge capable of 400 × g centrifugation
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Figures

Videos

Literature Cited

Literature Cited
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