5.10. Annexin-V apoptosis assay

BJ-5ta cells were seeded on six well plates at a density of 25,000 cells per well and incubated for 24 h. Cells were then transfected and incubated for 48 h in transfection media before this was replaced with treatment media containing either 25μM etoposide (Merck, E2600000) or an equivalent amount of DMSO, or in the case of cisplatin treatment media containing 2μM cisplatin or an equivalent amount of water. Cells were then incubated for a further 24 h and then cells were harvested with trypsin along with their media and PBS wash, pelleted at 300 g for 5 min, washed once in PBS and re-pelleted. Cells were then resuspended in 500 μL Annexin V binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl) supplemented with 1 u L Annexin V-FITC (ab14082) and 5 u L 7-AAD (BioLegend, 420403) and incubated for 10 min in the dark before scanning on an Attune NxT flow cytometer.