[17] Cytofluorometric quantitation of nuclear apoptosis induced in a cell-free system

Cytoplasmic extracts and mitochondrial intermembrane proteins contain factors that can induce apoptotic changes, including chromatin condensation and oligonucleosomal DNA fragmentation in isolated nuclei in vitro. These changes can be revealed by the agarose gel electrophoresis of DNA or by staining of nuclei with 4´, 6-diamidino-2-phenylindole dihydrochloride (DAPI) or other DNA-intercalating dyes, followed by the visual inspection of nuclei in a fluorescence microscope. This chapter discusses a method for the quantitation of nuclear apoptosis by the cytofluorometric analysis of propidium iodine-stained HeLa nuclei. Nuclei whose DNA is fragmented contain less DNA than control nuclei, and thus give rise to a sub-G0/1 peak. The frequency of hypoploid nuclei strictly correlates with that determined by morphological criteria. This method provides a highly reproducible and reliable method for the accurate and rapid quantitation of nuclear apoptosis induced in the cell-free system. Background values depend on the quality of HeLa cultures and on the duration of storage of nuclei. As in other biochemical assays, dose–response curves should be performed for each sample to allow for quantitative comparisons. This technique is superior to previous methods based on the visual inspection of isolated nuclei, because it provides quantitative data and allows for the analysis of hundreds of samples per day.