FACS on stromal cells ex vivo
spamcollector at tragoc.f2s.com
Thu Oct 20 18:06:21 EST 2005
Kyle Legate wrote:
> 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
>> 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?
> 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.
depends on the cytometer you have, a standard Facscalibur (or similar) won't
go down as far as DAPI. You could try some of the mitochondrial dyes as
I'd guess rbcs won't stain up as much (DIOC6 or the mol probes mitotracker
dyes might work). Of course if you're ust doing analysis and don't need
the cells out at the end, you could permeabilise them and stain with PI
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