G2/M interpretation
ZBIGNIEW DARZYNKIEWICZ (darzynk@nymc.edu)
Tue, 13 May 1997 17:24:18 -0500
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