Cell-cycle synchronization, flow cytometry and immunofluorescence
HeLa-EGFP-hOGG1 and HeLa-EGFP were synchronized at the G1/S boundary by two cycles of thymidine blockage and at the G2/M boundary by nocodazole treatment. Briefly, exponentially growing cells were blocked for 16 h in 2 mM thymidine containing DMEM, released for 9 h in thymidine-free DMEM and blocked again with 2 mM thymidine containing DMEM for another 16 h. Synchronization at G2/M was achieved by culturing the cells with nocodazole (40 ng/ml) for 16 h. After the double-thymidine block and the removal of nocodazole, the cells were allowed to progress under normal conditions and harvested at different time points after release. The cell-cycle phase distribution of a cell population was determined by measuring cell DNA contents by flow cytometry as follows: cells (1 × 106) were trypsinized, washed and fixed in 75% ice-cold ethanol and stored at 4°C. Immediately before flow cytometric analysis, the samples were treated with RNase A for 30 min at room temperature and then stained with propidium iodide (final concentration 50 μg/ml) for 30 min at 37°C. The samples were analysed by using a BD LSR flow cytometer (Becton Dickinson, NJ). For immunofluorescence, cells were grown on glass coverslips and fixed with 4% paraformaldehyde for 20 min at room temperature at different time points after release. Cellular DNA was stained with 0.25 μg/ml 4′,6′-diamino-2-phenylindole (DAPI) for 10 min at room temperature. Coverslips were mounted with fluorescent mounting medium (DAKO) and visualized using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Jena, Germany).