Nüsse M, Jülch M, Geido E, Bruno S, Di Vinci A, Giaretti W, Ruoss K
GSF-Institut für Biophysikalische Strahlenforschung, Frankfurt/M, Federal Republic of Germany.
Cytometry. 1989 May;10(3):312-9. doi: 10.1002/cyto.990100310.
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.
通过用溴脱氧尿苷（BrdUrd）对S期细胞进行脉冲标记，并对掺入BrdUrd的细胞和总DNA含量进行染色，可以很好地区分有丝分裂细胞与处于细胞周期G1期、S期和G2期的细胞。根据碘化丙啶荧光，未标记的G2期和M期细胞可作为两个单独的峰进行测量。与G2期细胞相比，M期细胞显示出较低的碘化丙啶荧光发射。M期和G2期细胞的荧光差异是由它们DNA的不同热变性引起的。在95℃热处理30 - 50分钟后，M期和G2期细胞得到了最佳分离。如果细胞培养物中不存在未标记的S期细胞，则可以测量有丝分裂指数。通过额外测量90度散射和/或前向散射信号，可以清楚地将有丝分裂细胞与未标记的G2期和S期细胞区分开来。通过对选择性分选后核形态的视觉分析，验证了从间期细胞中正确区分有丝分裂细胞（约99%）。随着脉冲标记的细胞在细胞周期中进展，可以观察到未标记和标记的有丝分裂细胞。我们得出结论，这种使用延长热变性和同时测量散射信号的改良BrdUrd/DNA技术可能会提供额外的信息，特别是在存在未标记BrdUrd的S期细胞的情况下。
