Annexin 5 binding buffer solution

For Cy5 Annexin V (BD Biosciences) and DAPI staining followed by flow cytometry analysis, equal numbers of N/TERT, SINCE and SINEG2 cells were seeded in 6-cm dishes, and collected either 24 or 48 h after plating, or were collected after 24 h of mock treatment or UVB treatment for 24 h in order to detect UVB-induced apoptosis. Culture media from each of UVB-treated culture dishes were also collected to include any detached dead or apoptotic cells that may be floating in the medium. After centrifugation, cells were resuspended in 100 μl of 1 × Annexin V-binding buffer solution (BD Biosciences) and 5 μl Cy5 Annexin V antibody (BD Biosciences) or without antibody (negative controls). Resuspended cells were then gently vortexed and were incubated for 15 min at room temperature in the dark. Next, 400 μl of 1 × Annexin V-binding buffer solution (BD Biosciences) was added per sample and samples were kept on ice. Finally, DAPI (Sigma-Aldrich) was added at a final concentration of 200 ng/ml per sample, 1–2 min before the cells were given for flow cytometry analysis, where the same number of events was acquired for each sample (20 000–30 000 events). Samples were analysed in duplicates.