Facs calibur cell flow cytometer

Cells were plated on six-well plates and were allowed to adhere. Following different treatments for 48 h, cells from each group (5×10⁵ cells/group re-suspended in 0.5 ml ice-cold RPMI 1640) were transferred into a clean centrifuge tube and incubated with 1.25 µl Hoechst 33342 for 15 min at room temperature in the dark to stain the nuclei of all cells. Subsequently, the plates were centrifuged at 2,000 × g for 5 min and the supernatants were discarded. Cells were then re-suspended in 0.5 ml ice-cold RPMI 1640 prior to the addition of 10 µl PI (5 µg/ml) for staining of the dead cells. The samples were kept on ice in the dark and immediately analyzed using a FACSCalibur Cell flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cytotoxicity was expressed as the percentage of dead cells (PI-positive) relative to the total number of cells.