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 e stimating apoptosis are based on biochemical, electrophoretic, or flow cytometry analysis of nuclear DNA. However, apoptosis is also associa ted with a chain of morphologic changes in the nuclear and cytoplasmic structures that are easily recognizable using light microscopy. We su ggest that changes in morphology of cells undergoing apoptosis might c ause characteristic changes in their optical properties. It follows th at 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 th e MiCK assay was employed to follow OD changes in HL-60 and OCI/AML-3 myelogenous leukemia cells and murine thymocytes exposed to ethanol, h ydrogen peroxide, etoposide, cisplatin, doxorubicin, or hyperthermia; i.e., diverse stimuli known to induce cell death via apoptosis or necr osis. The MiCK assay revealed prominent differences between the optica l 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 ''necroti c'' 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. Criteri a for graphical estimate of apoptosis were suggested and used to study apoptosis induced in HL-60 cells by the chemotherapy compounds etopos ide and cisplatin. The MiCK assay demonstrated markedly varying time c ourses of the cell apoptotic response to these two drugs. In both case s, however, the time of graphically predicted peaks of apoptosis corre lated with the time of morphologically and electrophoretically recogni zed peaks of apoptosis. Adaptability of the MiCK assay to a 96-well mi croplate format opens the way for large-scale studying of cell apoptot ic response to various stimuli. An important technical advantage of th e automated MiCK assay of apoptosis is that it does not require additi onal laboratory procedures after microcultures are initiated.