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Chapter 32 : Flow Cytometric Detection and Quantification of Apoptotic Cells

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Abstract:

Programmed cell death is a normal, physiological event which occurs without inflammation, so nontargeted bystander cells are not harmed. It is the body’s mechanism for eliminating cells which are no longer needed or potentially injurious. The study of apoptosis is being increasingly applied to many different cell types (e.g., hepatocytes, neurons, cardiac myocytes, etc.). The methods discussed in this chapter refer to the analysis of apoptotic cells in the sample of interest by utilizing flow cytometry, which has emerged as a technology well suited to quantifying dying cells. The subdiploid assay is highly recommended as an initial assay for a simple evaluation of apoptosis. The major advantages of this method include its ease, similarity to familiar assays of the cell cycle, and low cost, and in addition, following flow cytometric analysis, samples can be spun down onto slides and examined by fluorescence microscopy. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method is based on the enzyme-mediated insertion of nucleotides into DNA strand breaks which are present in apoptotic cells. Apoptotic cells containing active caspase-3 or -7 will exhibit an increased fluorescence intensity compared to that of the live cells in the same sample. The potential to treat human disease via manipulation of apoptotic pathways holds great promise, and as the elucidation of both protective and inductive cell death signaling mechanisms continues, such possibilities approach realization.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32

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Plasma Membrane
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Cellular Processes
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Flow Cytometric Assay
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Programmed Cell Death
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Image of FIGURE 1
FIGURE 1

Major mechanisms for regulation of cell death. Cell death pathways may involve (i) the interaction between a cell surface receptor capable of signaling for apoptosis and its ligand; (ii) DNA damage, after which chromosome repair or cell deletion mechanisms may commence; (iii) alterations in the mitochondria, which are major cellular organelles involved in the decision for replication or elimination; and (iv) an imbalance in apoptotic regulatory molecules, such as those in the Bcl-2 family.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 2
FIGURE 2

Rationale for subdiploid apoptosis assay. A hallmark of apoptosis is the activation of endonuclease, resulting in the fragmentation of cellular DNA. Samples are fixed and permeabilized with ethanol, which allows DNA fragments to escape past the plasma membrane. Staining with pro-pidium iodide (PI) and measuring the DNA content flow cytometrically reveal a population with reduced DNA content representing apoptotic cells. (Inset) Apoptotic cells are a distinct population with reduced fluorescence (shaded population).

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 3
FIGURE 3

Representative histogram of subdiploid method for assay of apoptosis. Peripheral blood mononuclear cells (PBMC) from an HIV-infected child were cultured for 3 days and then processed by the subdiploid assay. Cells with reduced DNA content compared to G cells are clearly visible.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 4
FIGURE 4

Rationale for TUNEL apoptosis assay. Endonuclease activation during apoptosis results in DNA strand breaks. Samples are fixed and permeabilized. The enzyme terminal deoxynucleotidyl transferase inserts fluorescently labeled nucleotides, allowing the resolution of apoptotic from live cells. (Inset) Apoptotic cells exhibit an increased fluorescence intensity compared to live cells (shaded population). FITC, fluorescein isothiocyanate.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 5
FIGURE 5

Representative histogram of TUNEL assay. PBMC from an HIV-infected child were cultured for 3 days, prepared by the TUNEL method, and analyzed flow cytometrically. Apoptotic cells exhibit a bright fluorescence intensity.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 6
FIGURE 6

Determination of DNA strand breaks within a specific subpopulation. PBMC from an HIV-infected child were cultured for 3 days. The sample was then labeled with an anti-CD4-phycoerythrin (PE) monoclonal antibody, fixed and per-meabilized in Ortho Permeafix, and then subjected to the TUNEL procedure. This experimental design allows the quantification of apoptotic CD4 cells.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 7
FIGURE 7

Rationale for annexin V apoptosis assay. In live cells, a state of membrane asymmetry exists in which phosphatidylserine (PS) is sequestered in the inner portion of the plasma membrane. During apoptosis, phosphatidylserine translocates to the outer cell membrane. Fluorochrome-conjugated annexin V binds specifically, thus tagging apoptotic cells. (Inset) Annexin-positive cells exhibit enhanced fluorescence compared to live cells (shaded population).

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 8
FIGURE 8

Representative histogram for detection of apoptosis by annexin method. PBMC from an HIV-infected child were cultured for 3 days, prepared by the annexin method, and analyzed by flow cytometry. Phosphatidylserine-expressing cells are distinguishable from live cells as a separate peak.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 9
FIGURE 9

Determination of phosphatidylserine exposure within a specific subpopulation. PBMC from a healthy adult were cultured for 3 days. The sample was labeled with anti-CD8-phycoerythrin-cyanine 5 (PE-CY5) and annexin fluorescein, washed, and fixed. Subsequent analysis allows the quantification of apoptosis within the CD8 subset.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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Image of FIGURE 10
FIGURE 10

Rationale for caspase apoptosis assay. A cell encounters an apoptosis-inducing stimulus, which initiates a cascade of intracellular events. Among these events in caspase-dependent apoptosis are the cleavage and activation of caspase enzymes. The fluorescent inhibitor of caspases (FLICA) is able to compete with the natural ligand and bind to activated caspase. (Inset) Cells can be analyzed by flow cytometry, and apoptotic cells will be detectable via their increased fluorescence.

Citation: McCloskey T, Phenix B, Pahwa S. 2006. Flow Cytometric Detection and Quantification of Apoptotic Cells, p 281-290. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch32
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