Lightscattering properties of apoptotic and necrotic cells

Intersection of a cell with the light of the laser beam in a flow cytometer leads to light scatter, and analysis of the signal of the scattered light reveals information about cell size and structure (34). The possibility of the analysis of light scattered in the forward and at right angles ('side scatter'; 90°) directions is a built-in feature of every commercially available flow cytometer utilizing laser illumination, and is a routine measurement, either alone or in conjunction with the measurement of cell fluorescence. Forward light scatter correlates well with cell size, whereas side scatter bears information on the cell light refractive and reflective properties and reveals optical inhomogene-ity of the cell structure, such as that resulting from condensation of the cytoplasm or nucleus, granularity, etc. Side scatter, however, cannot be measured by LSC.

As a consequence of cell shrinkage, a decrease in forward light scatter is observed at a relatively early stage of apoptosis (Figure 2). Initially, there is little change in side scatter during apoptosis, though in some cell systems an increase in intensity of the side scatter signal is seen, reflecting, perhaps, chromatin and cytoplasm condensation and nuclear fragmentation. When apoptosis is more advanced and the cells become small, the intensity of the side scatter decreases, just as the forward scatter. Late apoptotic cells are thus characterized by a markedly diminished intensity for both the forward and



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Figure2. Light scattering properties of apoptotic cells. To induce apoptosis HL60 cells were treated with CPT for 3 h (16, 17). Their light scattering properties were measured by FACScan (Becton Dickinson, San Jose, CA). The heterogeneity of apoptotic cells is reflected by the large variability in the scattered light. The early apoptotic cells (Ap) have diminished forward scatter but show no change in side scatter.The cells that are more advanced in apoptosis have both the forward and side scatter greatly decreased.

side scatter signals. In contrast to apoptosis, cell swelling, which occurs early during cell necrosis, is detected by a transient increase in forward light scatter. Rupture of the plasma membrane and leakage of the cytosol during subsequent steps of necrosis correlates with a marked decrease in the intensity of both forward and side scatter signals.

Because there is a great variability in the light scattering properties of individual cells, an exponential scale (logarithmic amplifiers) should be used during scatter measurements. Analysis of light scatter is often combined with other assays, most frequently with surface immunofluorescence (e.g. to identify the phenotype of the dying cell), or with another marker of apoptosis. It should be mentioned, however, that the light scatter changes alone are not a very specific marker of apoptosis or necrosis. Mechanically broken cells, isolated nuclei, cell debris, and individual apoptotic bodies may also have diminished light scattering properties. Therefore, the analysis of light scatter should be combined with measurements that can provide a more definite identification of apoptotic or necrotic cells.

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