Automated monitoring of apoptosis in suspension cell cultures.
Cell death by apoptosis is often accompanied by extensive DNA cleavage at internucleosomal linker sites. Thus, the foremost techniques for estimating apoptosis are based on biochemical, electrophoretic, or flow cytometry analysis of nuclear DNA. However, apoptosis is also associated with a chain of morphologic changes in the nuclear and cytoplasmic structures that are easily recognizable using light microscopy. We suggest that changes in morphology of cells undergoing apoptosis might cause characteristic changes in their optical properties. It follows that continuous measuring of the OD of cells undergoing apoptosis would enable the study of the kinetics of cell death. We recently described an automated microculture kinetic (MiCK) assay that provides multiple OD measurements in nondisrupted cell cultures. In the present study the MiCK assay was employed to follow OD changes in HL-60 and OCI/AML-3 myelogenous leukemia cells and murine thymocytes exposed to ethanol, hydrogen peroxide, etoposide, cisplatin, doxorubicin, or hyperthermia; i.e., divers stimuli known to induce cell death via apoptosis or necrosis. The MiCK assay revealed prominent differences between the optical properties of the cells undergoing the two different modes of death. Plotting the OD data accumulated during the assay period against time betrayed characteristic patterns of either "apoptotic" or "necrotic" OD curves. The best fit slope of the indicative of apoptosis steep rising component of the apoptotic curve, correlated directly with the percentage of morphologically apoptotic cells in the culture. Criteria for graphical estimate of apoptosis were suggested and used to study apoptosis induced in HL-60 cells by the chemotherapy compounds etoposide and cisplatin. The MiCK assay demonstrated markedly varying time courses of the cell apoptotic response to these two drugs. In both cases, however, the time of graphically predicted peaks of apoptosis correlated with the time of morphologically and electrophoretically recognized peaks of apoptosis. Adaptability of the MiCK assay to a 96-well microplate format opens the way for large-scale studying of cell apoptotic response to various stimuli. An important technical advantage of the automated MiCK assay of apoptosis is that it does not require additional laboratory procedures after microcultures are initiated.
Kravtsov
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Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.
Fabian
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I
D000818:Animals; D017209:Apoptosis; D001331:Automation; D002460:Cell Line; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology; D003584:Cytological Techniques; D004249:DNA Damage; D000431:Ethanol / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005260:Female; D018922:HL-60 Cells / Q000187:drug effects / Q000473:pathology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009336:Necrosis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013950:Thymus Gland / Q000166:cytology
D000431:Ethanol; D005047:Etoposide; D006861:Hydrogen Peroxide; D002945:Cisplatin