There isn't much in terms of counts in the supernatant and I do see a nice large pellet after spinning down the etOH. When I aspirate off the supernatant, I leave a really large buffer so I don't aspirate out any cells. The pellet looks intact after aspirating. After 1st wash in PBS, the pellet is about half the size. How am I losing these cells?
Here is a recap of my protocol:
Wash CHO cells, final conc 2-8E6 cells, in cold PBS twice (320xg 5min)
Add 0.75ml of PBS to pellet and add dropwise 70% etOH while the cells are vortexing on low
Put in freezer -20C overnight
Spin down cells in etOH at 700xg (I've even tried 850xg to no avail) for 5 min
Add 2 ml PBS and count
for the purpose of staining with propidium iodide and analyzing on a flow cytometer.
Edited by rosakim, 04 February 2013 - 04:10 PM.