Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements.
Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.
Nüsse
M
M
GSF-Institut für Biophysikalische Strahlenforschung, Frankfurt/M, Federal Republic of Germany.
Jülch
M
M
Geido
E
E
Bruno
S
S
Di Vinci
A
A
Giaretti
W
W
Ruoss
K
K
D000818:Animals; D001973:Bromodeoxyuridine; D002453:Cell Cycle; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D008938:Mitosis; D011419:Propidium; D013861:Thiocyanates
D005452:Fluoresceins; D013861:Thiocyanates; D011419:Propidium; D004247:DNA; D001973:Bromodeoxyuridine; D016650:Fluorescein-5-isothiocyanate