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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. |
