Flow cytometry for cell cycle and apoptosis analyses

Cells in 6-well plates were collected using tryptase 48 h following transfection. Cells were washed twice with PBS, followed by resuspension in 250 µl binding buffer (BD Pharmingen; BD Biosciences). Cells were fixed in 1% paraformaldehyde at 4°C overnight and then stained with 5 mg/ml propidium iodide (PI) alone or together with Annexin V/fluorescein isothiocyanate (BD Pharmingen; BD Biosciences) at room temperature for 15 min for cell cycle or apoptosis analysis, respectively. Incubation was performed in the dark for 15 min. Flow cytometry was performed using flow cytometer and analyzed using NovoExpress 1.2.5 software (ACEA Biosciences, Inc.; Agilent Technologies, Inc., Santa Clara, CA, USA). The apoptotic rate was calculated by adding the percentage of early apoptotic (Annexin V-positive, PI-negative) and late apoptotic cells (Annexin V-positive, PI-positive).