Flow cytometric cell cycle analysis

The effect of VA on MCF-7 and MDA-MB-231 cell cycle distribution was determined by flow cytometric analysis. Cells were seeded in six-well plates at 1.4×10⁵ cells per well, cultured for 24 h and synchronized by serum deprivation for overnight prior to the indicated concentrations of VA exposure for 24, 48 and 72 h respectively. The cells were detached from the plates by trypsinization and fixed in 70% cold ethanol (added in a drop-wise manner) for at least 2 h at 4°C. Prior to flow cytometric analysis, the cell solutions were centrifuged at 300g for 5 min and the pellet was re-suspended in 1 ml of PBS/1% FBS. After centrifuging at 300 g for 5 min, the fixed cells were then treated with 0.5 ml of RNase A (200 µg/ml) and incubated for 10 min at room temperature. The DNA content per cell was analyzed using flow cytometry (BD LSRFortessa™ cell analyzer, San Jose, CA, USA) after being stained with PI staining solution (2 µg/ml) for 20 min at room temperature in darkness. Offline analysis of cell cycle distribution was performed using Summit4.3 software (Beckman Coulter, Inc). Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. 10,000 events per sample were recorded for each experiment.