Cell Proliferation Assay by BrdU Staining

In order to avoid cell–cell-contact-induced inhibition of cell proliferation due to cell confluency, we seeded 3-5 × 10⁵ cells per well in 6 wells plate. This procedure ensured that cells were in a sub confluent state after 1 day and 3 days of incubation. 10 μM of 5-bromo-2′-deoxyruridine (BrdU, Sigma-Aldrich) was added to cell cultures for 1 hour. After 1 hour pulse, the cells were fixed in ice-cold 70% ethanol dropwise on a vortex and left at 4 °C for 30 minutes, washed twice with PBS and suspended in 2 M HCl for 30 minutes at room temperature (RT) with occasional mixing. The cells were subsequently washed twice in PBS and 2.5ul of anti-BrdU mAb (B44, BD biosciences) in BSA-PBS-Tween (PBS + 0.1% BSA + 0.2% Tween20, pH 7.4) were added to the cell pellet at RT for 30 minutes in the dark. After washing the cells twice, 2.5ul of goat anti-mouse FITC (BD555988, goat anti mouse) in PBS-Tween was added to the cells at RT for 30 minutes. The pellet was washed in PBS, 10μg/ml of RNAse (Ribonuclease A, R4642, Sigma-Aldrich) was added for 15 minutes at 37 °C and 50μg/ml of PI (Propidium Iodide, Sigma-Aldrich, #P4170) was added prior flow cytometry analysis. We collected at least 30,000 events at the BD FACS Canto II (BD Biosciences).