EtOH + PBS = precipitation?

Colin Rasmussen colin at fungus.biochem.ualberta.ca
Wed Feb 28 22:40:28 EST 1996


LU Pin wrote:
> 
> Colin Rasmussen (colin at fungus.biochem.ualberta.ca) wrote:
> : LU Pin wrote:

> Why prepare 95% ethanol + 5% PBS:
> I am analysizing the synchronization of mammalian cells by measuring the DNA
> contents using flow cytometer. I got a protocol from one of my colleages.
> Briefly, cells are washed in PBS, then fixed in 95% ethanol. Cells
> will be stained by propidium iodide subsequently. And I was told that the
> 95% ethanol should be prepared from abs. ethanol in PBS. That means 95%
> ethanol + 5% PBS. Then I have this problem. Now I prepare 95% ethanol in
> 160 mM NaCl. From the reply I got, it seems no way to avoid precipitation
> in 95% ethanol/PBS.

If you are preparing cells for FMF, that is something I have done. We usually wash 
the cells in Hank's Buffered Saline, resuspend in HBSS then fix by slowly adding 
ethanol to a final concentration of 70% EtOH. After that we go through the RNase 
treatment and staining with PI. If you want I can send you a detailed protocol. 
Works great everytime.

Colin



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