Annexin binding buffer

One hundred fifty thousand cells (SW-1710) and two hundred thousand cells (T-24) per well of a six well plate were seeded and reversely transfected with XtremeGENE™ HP (Roche). After 16 h cells were split into two wells. 48 h post transfection, supernatant and cells were collected, centrifuged at 1000 rpm for 5 min, washed with ice-cold 1x Annexin binding buffer (Serva, Heidelberg, Germany) and centrifuged again. The pellet was resuspended in 75 μl 1x Annexin binding buffer containing 4.5 μl Annexin V-APC (Serva) and 7.5 μl PI (1 mg/ml, Serva) and incubated for 15 min in the dark at RT. The suspension was diluted with 500 μl 1x Annexin binding buffer, centrifuged, washed and fixed with 0.5% methanol-free formaldehyde for 20 min on ice. The reaction was stopped with 500 μl 1x Annexin binding buffer. FACS measurements and analysis was performed using the MACSQuant Analyzer X and MACSQuantify Software (Miltenyi Biotec, Bergisch-Gladbach, Germany). In total, 50,000 cells/experiment were analyzed in three independent experiments.