Facscanto cell cytometer

Apoptosis was assessed by Annexin V/Propidium Iodide (PI) staining (IMMUNOSTEP, Spain) [43 (link)]. FACS analysis was performed using a FACSCanto™Cell Cytometer and the FACSDiva™ software (Becton-Dickinson, Italy).

Apoptosis was assessed by Annexin V/Propidium Iodide staining according to the manufacturers' instructions (BD Pharmingen). After 24 hours of treatment cells were labeled with FITC-conjugated Annexin V and Propidium Iodide (Becton-Dickinson, Italy). Fluorescence Activated Cell Sorting (FACS) analysis was performed using a FACS-Canto Cell Cytometer and the CellQuest software (Becton-Dickinson, Italy).

Cells were treated with DMSO [0.1%] or SB225002 with the following concentrations: REH and RS4;11 [10 μM]; Jurkat and TALL-1 [3.125 μM]. After 24 h of treatment, cells were centrifuged and fixed in cold 70% ethanol, washed twice with PBS and stained with 1 ml of propidium iodide solution (50 μg/ml propidium iodide, 3.8 mM sodium citrate in PBS) supplemented with 50 μl RNase A (50 mg/ml) for 1 h at room temperature and analyzed with a FACSCanto cell cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). At least 20,000 cells were collected and the cell cycle profiles were calculated using the BDFACSDiva (Becton Dickinson).