Cell recovery from ethanol fixation - (Feb/04/2013 )

After ethanol fixing CHO cells overnight and spinning them down at 700-850 x g, I am losing about half of my cells. A part of the reason I may be losing cells is from cells aggregating and clogging the aperture on an impedence based cell counter. But if this was solely the case, I would not be able to get a consistent cell count each time. To avoid cell aggregation, I first add 0.75ml of PBS to the pellet and pipette triturate well before adding the 70% etOH dropwise.
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.

I put EtOH in -20 or -80 freezer for at least 1 hr before I resuspend my cell pellet in it. I think 750 ul PBS is a lot. I don't even centrifuge my cells after addition of EtOH. The Flowcytometer will do the counting for me.

Sorry, I did forget to mention that the 70% ethanol was in the freezer prior to use.
Really? You analyze without removing the ethanol? I didn't know that was possible.
I am looking at cell cycle arrest.

I would try with a slower spin for a longer time. Your fixed cells are likely very fragile and your fast spin may be damaging them, resulting in your decreased recovery.

Depends, i do remove ethanol if i want to do a PI test.