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And you're right that the cells need to be fixed first. I recommend fixing
for the exact time suggested in the protocol, longer (even a few minutes)
greatly limits the staining, probably by deterioration of the proteins. We
also use Fc-block from Pharmingen as a first step in staining, which
limits the background staining. Just to make clear, we use the exact
staining protocol from Pharmingen without modification!
The important variables are the frequency of antigen-responsive cells in
your mice/culture, and using monensin (or brefeldin a) to allow the
cytokine to build up inside the cell. We get very good staining with
anti-IFNg, decent staining with anti-IL4 (less bright, and also more prone
to high non-specific staining without Fc block), good staining with
anti-IL10, and not very much anti-IL12 staining (maybe little production
in our cultures).
When we first started we practiced with superantigen (SEB) stimulation of
murine LN cells to be sure we were getting stimulation of enough cells.
That worked great for us and allowed us to practice the protocol.
Good luck!
Lisa Glickstein, Ph.D.
Instructor of Medicine
New England Medical Center
Boston, MA 02111
On Thu, 3 Apr 1997, Richard K. Meister wrote:
>
> Hello, all:
>
> We are interested in counting the number of cells expressing various
> cytokines in a mouse model using flow cytometry. The only published work
> I've seen looking at intracellular cytokines and flow was in vitro
> stimulation in the presence of a protein transport inhibitor.
>
> Do any of you have experience detecting intracellular cytokines from in vivo
> experimental sytems (such as mouse blood or spleen cells) by flow? Is it
> reasonable to expect to be able to get anti-cytokine antibody molecules into
> a cell without the cytokines leaking out? (I assume a fixation step would
> have to preceed the permeablization step.) I'd appreciate any information
> about, or directions to, a protocol which works.
>
> Thanks in advance.
> * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
> * Richard K. Meister Email: Meister.1@osu.edu *
> * The Ohio State University Voice: (614) 292-9716 *
> * Dept. of Veterinary Biosciences FAX: (614) 292-6473 *
> * Cytometry Instrumentation Lab *
> * 1925 Coffey Road *
> * Columbus, OH 43210 U.S.A. *
> * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
>
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
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If you have any comments please direct them to
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