FACS on stromal cells ex vivo

Kyle Legate legatek at hotmail.com
Tue Oct 18 14:46:53 EST 2005

Ian A. York wrote:
> I want to look at non-lymphoid cells, by flow cytometry, as close as 
> possible to the mouse -- i.e. without culturing the cells in the interim. 
> The big problem is generating single-cell suspensions and getting rid of 
> RBCs.  I've tried mincing various tissues (heart, liver, lung, kidney, and 
> ear and tail for fibroblasts) then incubating in collagenase for various 
> times.  This does seem to produce at least a few dispersed cells, but then 
> when I try to eliminate RBCs using ACK buffer (the same protocol we 
> routinely use for spleens) the treatment seems to be both either too 
> gentle and leave the RBC intact, or else too harsh and to kill the cells 
> of interest.  Or perhaps the collagenase treatment itself is too harsh.
> Can anyone point me to a protocol for producing stromal cells ex vivo, 
> suitable for FACS?  
> Thanks.
> Ian 
Since you're doing FACS already, once you have solved the problem of 
generating a single cell suspension, sort the cells with dapi. As RBCs 
have no nucleus, they won't retain it.

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