Introduction

Flow cytometry, by providing the possibility of rapid, accurate, and unbiased measurements of a variety of cell components on a cell by cell basis, has become an indispensable methodology in the analysis of cell death (see reviews in refs 1-6). One area of application of flow cytometry is in studies of the mechanism of cell death. In this application, flow cytometry is primarily used to immunocytochemically detect and measure the cellular levels of proteins such as members of the Bcl-2 family, proto-oncogenes c-myc or ras, tumour suppressor genes p53, pRB, and other molecules that play a role in cell death. It is also used in studies of cell function, particularly mitochondrial metabolism, which is closely associated with mechanisms regulating cell sensitivity to apoptosis (e.g. 7, 8). The major virtue of flow cytometry in this application is that it offers the possibility of multiparametric analysis of a multitude of cell attributes. This allows one to study the mutual relationship between the measured constituents. When one of the measured attributes is cellular DNA content, a relationship of other parameters to the cell cycle position or DNA ploidy is analysed. Because individual cells are measured the intercellular variability can be assessed, cell subpopulations identified, and rare cells detected.

Another area of application of flow cytometry is in the identification and quantitation of apoptotic or necrotic cells. Their recognition is generally based on the presence of a particular biochemical or molecular marker that is characteristic for either apoptosis or necrosis. A variety of methods have been developed, especially for identification of apoptotic cells. The apoptosis-associated changes in cell size and granularity can be detected by analysis of laser light scattered by the cell in forward and side directions (9). Some of the methods rely on the apoptosis-associated changes in the distribution of plasma membrane phospholipids (5,10). Others measure the transport function of the plasma membrane. Still other methods probe the mitochondrial transmembrane potential, which decreases early during apoptosis (7, 8, 11). Endonucleolytic DNA degradation that results in extraction of low MW DNA from the cell provides yet another marker of apoptosis. Apoptotic cells are then recognized either by their fractional DNA content (12,13) or by the presence of DNA strand breaks which can be detected by labelling their 3'-OH ends with fluorochrome-conjugated nucleotides in a reaction utilizing exogenous terminal deoxynucleotidyl transferase (TdT) (14-18).

The common drawback of flow cytometric methods is that the identification of apoptotic or necrotic cells relies on a single parameter reflecting a change in a biochemical or molecular feature of the cell that is assumed to represent apoptosis or necrosis. However, such a feature may be absent when apoptosis is atypical, as is known to occur, e.g. in cells of epithelial and fibroblast lineage (19-21). Atypical apoptosis is also caused by agents that inhibit apoptotic effectors. For example, induction of apoptosis by an inhibitor of the endo-nuclease results in a lack of DNA fragmentation, while inhibitors of proteases prevent degradation of particular proteins such as 'death substrates' (e.g. ref. 22). Identification of apoptotic cells based on the missing attribute(s) (e.g. DNA fragmentation or degradation of a particular protein) is impossible in such cases. Therefore, the characteristic changes in cell morphology, as originally described (23, 24) and discussed in Chapter 2, still remain the gold standard for recognition of apoptotic cell death.

The laser-scanning cytometer (CompuCyte, Cambridge, MA) is a microscope-based cytofluorometer that offers advantages of both flow cytometry and image analysis (for reviews see refs 25 and 26). Thus, fluorescence of individual cells is measured rapidly by laser-scanning cytometry (LSC) and with an accuracy similar to that of flow cytometry. Since cell position on the slide is recorded together with other measured cell parameters in a listmode fashion, the cells can be relocated after measurements and re-examined visually or subjected to image analysis (e.g. 27, 28). Furthermore, the geometry of cells attached to the slide, especially when flattened by cytocen-trifugation, is more favourable for their morphometric analysis than when in suspension. More information on cell morphology, therefore, can be obtained by LSC than by flow cytometry. In the analysis of apoptosis and necrosis, an opportunity to examine measured cells visually, as offered by LSC, is of particular value. Furthermore, cell analysis on slides eliminates cell loss, which generally occurs during repeated centrifugations in sample preparation for flow cytometry.

Several flow cytometric methods developed for the identification of apoptotic and necrotic cells have been modified and adapted so that they may be used for LSC (e.g. 29-31). These modifications and changes in methodology required by the adaptation of flow cytometric methods to LSC are presented in this chapter. Also discussed are difficulties in the measurement of apoptosis or necrosis, as well as common errors in the analysis and interpretation of data.

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