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FACS on stromal cells ex vivo

FACS on stromal cells ex vivo - Protocols and Methods Forum

FACS on stromal cells ex vivo - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-18-2005, 05:51 PM
Ian A. York
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Default FACS on stromal cells ex vivo




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
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very respectable Man." -Jane Austen, The History of England
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  #2  
Old 10-18-2005, 07:25 PM
Nick Theodorakis
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Default FACS on stromal cells ex vivo


Ian A. York wrote:

Have you tried to get rid of the RBCs by Ficoll gradient? They are
pretty dense and pellet through Ficoll (as also do PMNs), whereas most
mononuclear cells will not.

Nick

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  #3  
Old 10-18-2005, 07:33 PM
Ian A. York
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Default FACS on stromal cells ex vivo

In article <1129663500.476748.23050@g43g2000cwa.googlegroups. com>,
Nick Theodorakis <[Only registered users see links. ]> wrote:

Haven't tried that. Do you have a protocol and/or reference handy?

Ian

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Ian York ([Only registered users see links. ]) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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  #4  
Old 10-18-2005, 07:46 PM
Kyle Legate
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Default FACS on stromal cells ex vivo

Ian A. York wrote:
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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  #5  
Old 10-20-2005, 06:42 PM
Nick Theodorakis
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Default FACS on stromal cells ex vivo

Ian A. York wrote:

It's been many years since I did one, and I couldn't find it in my old
protocol books, but it's an established technique. You need some
material that is called Ficoll/Hypaque, but at least one manufacturer
calls it Histopaque. It comes as a premade solution. We used to just
layer about 20 ml in a 50 ml conical tube, layer the blood (or cell
suspension) on top, and spin at a few hundred g's. The red cells and
other dense cells like PMNs would pellet, and the PBLs would form a
layer. You could probably find more detailed instructions in a
hematology or histology text, or it just might come along with the
Histopaque if you buy some.

HTH,

Nick

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  #6  
Old 10-20-2005, 11:06 PM
Clive Tregaskes
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Default FACS on stromal cells ex vivo

Kyle Legate wrote:



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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