The many characteristics of apoptosis can be exploited to identify apoptotic cells amongst viable cell populations. The loss of DNA from cells as a result of DNA fragmentation is technically the easiest to detect. Small fragments consisting of 182-bp DNA multimers can be eluted from apoptotic cells after permea-bilization with 70% ethanol and washing in phosphate buffer. The permeabilization also allows entry of the dye PI into cells which intercalates with DNA. The resulting DNA profile for proliferating viable cells shows a DNA distribution for cells in G0/G1 phase, S-phase, and G2/M phase of the cell cycle, but the elution of DNA from apoptotic cells gives a population of cells with a DNA content less than that of cells in G0/G1.
The nucleosomal fragmentation of DNA starts with the initial formation of 300-kbp and/or 55-kbp fragments. It is only after this primary stage that the 182-bp fragments are generated. In some cell types, this latter fragmentation does not occur. These large fragments are too big to be eluted from cells, so on
Fig. 1. Apoptosis detection by YO-PRO-1 (alterations in cell plasma membrane). Live cells (L) are negative for both YO-PRO-1 and PI. Early apoptosis (A) shows increased YO-PRO-1 fluorescence but remains PI-negative. Dead cells (D) are YO- PRO-1- and PI-bright, but as the cells become necrotic, DNA degrades and the fluorescence of both dyes is reduced (N).
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