From: David.C.McFarland@gsk.com
Date: Mon Feb 24 2003 - 16:25:06 EST
Inna,
We were experiencing the same difficulties you describe with cell-cycle
analysis on rat embryonic vascular endothelial cells and also mouse stem
cells. We tried a number of different fix and/or perm strategies. What
worked for us was the technique described by Schmid, et al in: Measurement
of lymphocyte subset proliferation by three-color immunofluorescence and
DNA flow cytometry, J. Immun. Meth. V235, 2000, pp121-31. Basically, they
used a low pH phosphate-citrate buffer containing saponin; no fixation.
The cells remained intact and we got CVs comparable to PI staining using
their 7AAD/AD technique. I now recommend this to anyone who is working
with fragile cells.
BTW, what Abs are you using to label HPAECs?
David McFarland
GlaxoSmithKline
----- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 24-Feb-2003 16:08
-----
Inna.Cohen@med.va.gov
21-Feb-2003 08:56
To: "Cytometry Mailing List"
cc:
Subject: [Losing HPAECs during prep]
Dear Flow-ers,
We have a problem. We are trying to analyze HPAEC's(Human Pulmonary
Endothelial Cells) using flow cytometry. We are looking at cell cycle
distribution after hypoxia. Our problem is that most of our cells are
destroyed before we can analyze them. We batch them in ethanol and then
use
the high salt extraction method but most of the cells are simply blown
away
by the time we analyze them. So we tried using the Cell Cycle kit from
B-D
which uses detergent to break open the cells but we get the same results.
The cells just seem to burst before we can analyze them. Does anyone have
any suggestions? If anyone else out there in flow land is working with
these kinds of cells, any input would be greatly appreciated.
Thank you,
Inna
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