I am handling HL60 cells and am intending to stain with PI to study cell cycle. However, I am unable to obtain a pellet from the 2nd PBS wash after ethanol fixation.

1. What has gone wrong?

2. Does ethanol need to be chilled to -20°C? Does it make a huge difference? Saw 1-2 protocols that pre-chilled ethanol while troubleshooting.

3. Saw a previous thread that cell pellet was obtained after washing with FACS buffer instead? Would addition of FBS in the washing step help?

Here was what I did:

Cells were harvested and washed with 1 mL 1x PBS. Supernatant was removed. Pellet was briefly vortexed pdior to dropwise addition of 70% ethanol. While adding ethanol, the tube was vortexed.

The cells were fixed for 30 min on ice. Afterwards, cells were pelleted after 1500 g centrifugation for 5 min. Cells were washed with 1x PBS. Did not obtain a pellet after centrifugation at 500g for 8 min, then proceeded with 1000g for 5 min and 1500 g for 5 min. I was then advised by my RF to resuspend the solution and leave on bench for 5 min. Pellet was obtained after centrifugation with 1300g for 5 min.

Most upernatant was removed, leaving 50 uL. 2nd 1 mL 1x PBS wash was performed and cells were incubated for 5 min on the bench. Next, cells were then centrifuged at 1300g for 5 min. No pellet was ontained. Proceeded to resuspend the solution again and left on the bench for 5 min prior to centrifugation at 1500g for 10 min. No pellet was obtained.

FYI: 70% ethanol was prepared with analytical grade absolute ethanol & milliQ water. It was stored at 4°C prior to fixation.