Cell Cycle Analysis by FACS

For cell cycle analysis by FACS, cells suspended in Deitch buffer (10 mM Tris-hydrochloride (pH 7.5) / 5 mM MgCl₂) and stained with 100 µg/ml propidium iodide⁴⁴ were analyzed in a Coulter EPICS XL flow cytometer using SYSTEM II Version 3.0 software (Beckman-Coulter, Krefeld, Germany). The percentage of cells present in the sub-G1 peak, representing apoptotic cells, was calculated after exclusion of cell doublets. The sum of cells in G2 and S phase was defined as the percentage of proliferating cells. Alternatively, proliferating cells and pyknotic nuclei were counted manually from BrdU- and 4',6-Diamidino-2-phenylindole (DAPI)-stained cells on coverslips, respectively. For this purpose, in a survey, at least 10 different sections of one coverslip and at least 1000 cells were counted and the number of apoptotic, clearly pyknotic nuclei (at least 10) or clearly BrDU-positive stained cells was determined.