John Tonkinson inquires about the method to measure duration of G2. The simplest approach is to include BrdU into the culture and collect cells every every 30 or 20 min. The cells are smeared or cytocentrifuged on slides. Cellular DNA is then denatured by acid or heat and the incorporated BrdU stained with the FITC conjugated MoAbs. By UV light microscopy only mitotic cells are then analyzed. During the first hour or two after addition of the precuror all mitotic cells remain unlabeled. The time when the first BrdU labeled mitotic cell is observed represents the minimum duration of G2 for this cell population. The time when nearly all mitotic cells are labeled represents maximal duration of G2 for this cell population, and likewise when 50% mitotic cells are labeled is the modal duration of G2. One can, of course, be more sophisticated and use multiparameter flow cytometry combining detection of mitotic cells (e.g. based on absence of cyclin A and G2/M DNA content or some other feature) and BrdU incorporation, or using laser scanning cytometer to identify apoptotic cells, e.g as recently described by Gorczyca et al., (Cell Proliferation, 29: 539-548, 1996). Zbigniew Darzynkiewicz
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