Analysis of apoptotic cells by flow and laser scanning cytometry

A large number of flow cytometric methods to identify apoptotic cells and analyze morphological, biochemical, and molecular changes that occur during apoptosis have been developed. These methods are also applicable to the laser scanning cytometer (LSC), a microscope-based cytofluorometer that combines advantages of flow and image cytometry and that, by offering a possibility of assessment of cell morphology, is of particular utility in analysis of apoptosis. Apoptosis-related changes in cell morphology associated with cell shrinkage and condensation of cytoplasm and chromatin are detected by measurements of the intensity of light scatter of the laser beam in the forward and 90 degrees angle directions. Changes in plasma membrane composition and function are analyzed by its altered permeability to certain dyes and by the appearance of phosphatidylserine, which reacts with annexin V-fluorochrome conjugates on the external surface of the membrane. Decrease in mitochondrial transmembrane potential is measured with several fluorochromes of the rhodamine or carbocyanine family. DNA fragmentation is detected either by measurement of cellular DNA content after elution of the degraded DNA from the cell before or during the staining procedure or by in situ labeling DNA strand breaks. Apoptotic cells are then recognized either on the basis of their reduced DNA-associated fluorescence as the cells with fractional DNA content ("sub-G1 cells"), or as the cells with an extensive number of DNA breaks, respectively. Advantages and limitations of the preceding methods are discussed and their adaptation to LSC is presented.