A MULTIPARAMETER APPROACH TO CELL CYCLE ANALYSIS AS A STANDARD TOOL IN ONCOLOGY DRUG DISCOVERY - Flow Cytometry in Drug Discovery and Development - page 108

FIGURE 5.10 Control cells (a) and progressive stages of apoptosis (b) to (d). (See the color
version of the figure in the Color Plates section.)
but, in reality the cells disappear after a sequence of events that need a metabolically
active cell to produce the sequel of events. These events generally include DNA
cleavage and formation of small vesicles that will bleb from the surface of the cells
until there is no more cell to produce them. These blebs or vesicles will eventually
disappear by either secondary necrosis (in vitro) (see Figure 5.10) or by being
engulfed by phagocytic cells (in vivo) [19].
There are several technical approaches available to evaluate the different steps
occurring in apoptosis. The initiation step exposes in cells the phosphatidyl serine
that can be identified by annexin V signaling and progression can be evaluated by
intracellular cytochrome c and caspase 3 release, anti-PARP p85 fragment pAb
production, and DNA cleavage [20-23]. All are reported to be effective ap-
proaches for evaluating apoptosis. Gorczyca et al. also published a comparison of
these assays and their efficacy as an evaluation tool [24]. Each technical approach
has advantages and limitations due to the very active movements of the population
distribution of cells engaging in apoptosis across experimental times. Since cells
in culture (or, by the way, also in vivo) are never growing in synchronized waves,
apoptosis seldom occurs simultaneously in all cells at any given time and while
some might have already advanced to bleb into small vesicles or ultimately
disappeared under secondary necrosis, other cells in the same culture might have