Cell synchronization and flow cytometry analysis

For synchronization in G0, cells were washed 3 times in phosphate buffered saline (PBS) and serum deprived for 72 hours. To synchronize cells in G1, serum deprived cells were re-stimulated by adding culture media supplemented with 5% FBS for 6 hours. Synchronization at the G1/S transition by double thymidine block was performed by treating the cells with 2 mM thymidine for 16 hours. After the first block, the thymidine was washed out and replaced with fresh culture media for 8 hours. Thymidine (2 mM) was added back for 16 hours for a second block. To obtain a G2/M population, cells were treated with 100 ng/ml of nocodazole for 16 hours. Cell cycle progression was assayed by determination of DNA content using propidium iodide (PI) and bromodeoxyuridine (BrdU) labeling followed by flow cytometry analysis. BrdU was added for 30 min, and cells were harvested and fixed with 70% ethanol overnight. Cells were centrifuged at 10,000 x g for 1 min and washed in BrdU wash solution (0.5% Tween 20, 0.5% bovine serum albumin (BSA) in PBS). Cells were resuspended in 2 N HCl for 20 min at room temperature, neutralized with 0.1 M sodium borate, and washed twice with BrdU wash solution. Cells were incubated with anti-BrdU antibody (BD 347580 mouse IgG) at room temperature for 30 min. Cells were washed 3 times in BrdU wash solution and incubated with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) for 30 min at room temperature. Cells were washed 3 times in BrdU wash solution and then treated with 100 ug/ml RNase and 25 ug/ml propidium iodide. Analyses were performed with a BD LSRFortessa instrument.