Scott, Yalin and anyone else interested:
I scratched my head the first time I heard of this protocol as well but, I
have used a similar method and no RNase is necessary. As I understand it,
the low pH of the citrate buffer is sufficient to denature the RNA. It is
a totally alternative procedure to the traditional EtOH/MeOH fix and/or
fix&perm procedure. Using 7AAD/AD and saponin as described in the
reference below, I got very nice histograms. I would think PI would give
comparable results.
See:
Measurement of lymphocyte subset proliferation by three-color
immunofluorescence and DNA flow cytometry. Ingrid Schmid, Steve W. Cole,
Jerome A. Zack and Janis V. Giorgi. J. of Immunological Methods. 235:1-2:121-31. 2000.
Good luck,
David McFarland
GlaxoSmithKline
----- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 27-Mar-2002 20:04
-----
"Scott Tighe" <stighe@zoo.uvm.edu>
26-Mar-2002 16:46
To: "Cytometry Mailing List"
cc:
Subject: Re: a question on cell cycle analysis with PI
Yalin:
0.1% triton X will certainly make the cells permeable but will not fix
them. If you do not include RNase, then RNA will also be stained, which
will influence your DNA cell cycle data. If you choose to add RNase A,
use at a final concentration of 0.25 mg/ml in your samples. You can take
a look at our protocol if you wish; it is for general DNA cell cycle
staining of mammalian cells. Our protocol employs the use of ethanol
fix/perm, but certainly can be used with Triton X.
http://www.vermontcancer.org/Research/Cores/FlowProtocols.html
Sincerely
Scott Tighe
Vermont Cancer Center
Flow Cytometry Core Lab
Yalin Guo wrote:
>
> I am doing a cell cycle analysis using cultured and non-cultured
> human CD34+ cells. I got a protocol with PI staining (0.1% Na
> Citrate, 0.1% Triton X-100, 20 ug/ml PI), but without fixation
> and RNase. I should be very grateful to get any suggestions for
> this method.
>
> Yalin
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