When all tubes are incubated at ~1 10x6 cells/ml
- transfer suspension to FACS tube
- spin at 1250rpm @ 0 degrees C for 5 minutes then tip out supernatant
- resuspend in 1ml PBS (10x6 cells/ml)
- add 3ml 95% ethanol - mix with repeated passes through a pipette
- store overnight (or at least 1 hour)
- wash 2 times with PBS (4ml volume) - resuspend in remaining supernatant
after final wash
- add 1760ml PBS; 200uL stock PI solution (400ug/ml); 20uL RNAse stock (20mg/ml)
- leave at least 30 minutes at room temperature in dark
- analyse on cytometer.
(Mix the stain to make 1-2 ml of stain per tube)