Cell apoptosis measurements and cell cycle arrest analysis

Cells were grown to 80–90% confluence prior to FACS analysis. The cells were trypsinized, washed twice with FACS buffer (1X PBS containing 1% BSA and 5 mM EDTA), and re-suspended in the same buffer to a concentration of 10 x 10⁵ cells/100 μL. For cell apoptosis measurements cells were stained with Annexin-V-FITC antibody (Thermo Fisher) following flow cytometry using a Guava EasyCyte flow cytometer (EMD Millipore, Billerica, MA). FITC labeled IgG isotope control was used as a negative control. For cell cycle analysis cells were permeabilized with 2% paraformaldehyde and stained with PI. Cells were then fixed with 1% PFA/1X PBS and analyzed by flow cytometry using a Guava EasyCyte flow cytometer (EMD Millipore, Billerica, MA). Side scatter and forward scatter profiles were used to eliminate cell doublets.