Cell Cycle Analysis by Flow Cytometry

For cell cycle analysis, cells were trypsinized, washed twice with ice-cold PBS (100 × g for 5 min at 4°C), fixed with ice cold 70% ethanol and kept at 4°C until analysis. Fixed cells were centrifuged (200 × g for 5 min at 4°C), washed with PBS containing 0.05% Tween-20, and then resuspended in PBS containing 0.05% Tween-20, 10 µg/ml propidium iodide and 0.1 mg/ml RNase A for 1 h at room temperature. Analysis was performed using the Attune NxT Flow Cytometer (Life Technologies, Paisley, UK). Whole-cell extracts were prepared, separated by SDS-PAGE electrophoresis, and analyzed by quantitative immunoblotting using the Odyssey image analysis system (Li-cor Biosciences, Cambridge, UK), as previously described (30 (link), 31 (link)).