Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90° and forward scatter measurements

Abstract

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 bromodeoxyuridine (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°C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90° 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 pulselabeled 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 BrdUrdunlabeled S-phase cells.