4.4. Annexin V assay

CM or PCM were removed from both cell lines, and cells were washed with PBS. Non-adherent cells present in media were collected by centrifugation (1 000 rpm for 5 min). Trypsin EDTA (1 ml) was used to detach adherent cells. These were combined with the non-adherent cells to which 5 ml PBS was added, and the suspension was centrifuged as before to pellet the cells. Annexin V binding buffer (BD Biosciences, USA) (1–2 ml) was added to the cell pellet, and 100 µl of cell suspension was stained with Annexin V fluorescein isothiocyanate (FITC) antibody and propidium iodide (PI) (BD Biosciences, USA) in the dark for 15 min. Binding buffer was added (200 µl), the sample was briefly vortexed, and cells were acquired on the BD LSR II flow cytometer (BD Biosciences, USA). Testing was conducted in duplicate. The percentage of viable cells was determined by how many cells excluded PI staining.