Sub-G1 FACS analysis - basic question (Nov/24/2006 )

Hello everybody!
I found the following protocol in a publication.
Sub-G1FACS Analysis forApoptosis
[...] Forty-eight hours later, cells were trypsinized, collected by centrifugation, resuspended in PBS, and fixed in 70% ethanol at -20°C for 1 day. After centrifugation, the cells were washed in PBS and resuspended in potassium iodide (PI) staining solution (PBS, PI, RNase) (Boehringer Mannheim Antisense bcl-xl 546 and Chemotherapy Co., Indianapolis, IN). Specimens were incubated in the dark for 30 min at 37jC and then analyzed with the use of an EPICS Profile II flow cytometer (Coulter Corp., Hialeah, FL). All experiments were performed in triplicate.

And there is one (stupid?) question I need to ask:
"when they say resuspend in PBS and fix in 70% EtOH"...what do they mean? Add EtOH to the cell suspension (in PBS)? Or add EtOH to the cell pellet? And -of course- how much EtOH am I supposed to add??
Thanks in advance for your answers!

I believe is wash once with PBS, centrifuge and resuspend the pellet in 70% ethanol (vortexing so the cells dont form clumps)
Maybe can also be resuspend the cells in 300 ul pbs and add 700 ul of absolute ethanol for a final of 100 but it is the first one the one we use

Hello everybody!
I found the following protocol in a publication.
Sub-G1FACS Analysis forApoptosis
[...] Forty-eight hours later, cells were trypsinized, collected by centrifugation, resuspended in PBS, and fixed in 70% ethanol at -20°C for 1 day. After centrifugation, the cells were washed in PBS and resuspended in potassium iodide (PI) staining solution (PBS, PI, RNase) (Boehringer Mannheim Antisense bcl-xl 546 and Chemotherapy Co., Indianapolis, IN). Specimens were incubated in the dark for 30 min at 37jC and then analyzed with the use of an EPICS Profile II flow cytometer (Coulter Corp., Hialeah, FL). All experiments were performed in triplicate.

And there is one (stupid?) question I need to ask:
"when they say resuspend in PBS and fix in 70% EtOH"...what do they mean? Add EtOH to the cell suspension (in PBS)? Or add EtOH to the cell pellet? And -of course- how much EtOH am I supposed to add??
Thanks in advance for your answers!

Noooooooooo!!
You should wash your cells with PBS, pellet, resuspend in PBS (500ul for half a million cells), then put it on the shaker and add EtOH 70%, drop by drop, 3 times 500ul. If you resuspend in EtOH the cells will attach together and you have to avoid cell clumps. This is also the reason why you should vortex while adding EtOH... The cells can be left in EtOH ON or longer at 4 degrees or at -20 but the fixation can be done at -20 for 1hour and it works anyway

Watch out, final concentration of ethanol should be 70%, use absolute ethanol if you want to add 3 volumes of ethanol per volume of cells