Apoptosis and Cell Cycle Analysis

Cells were washed with PBS, then stained (10⁶ cells) in buffer containing FITC-annexin V and 7-amino-actinomycin D (7-AAD) (Beckmann Coulter, Fullerton, CA, USA) for 15 min at 4 °C and analyzed by flow cytometry (Becton Dickinson Accuri™ C6 flow cytometer). For cell cycle analysis [45 (link)], cells were first incubated with fixing solution (PFA 2%, Hepes 1%, saponin 0.03%) for 15 min and then in PFS permeabilization solution (PBS 1×, SVF 10%, saponin 0.03%, Hepes 1%). Cells were next stained for 30 min at room temperature with anti-Ki67-Alexa Fluor 488 monoclonal antibody or the corresponding isotype as control (Becton-Dickinson, Franklin Lakes, NJ, USA) before analysis by flow cytometry (Becton Dickinson Accuri™ C6 flow cytometer). The FlowJo^® software (V10.1, BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze data.