We've used this technique, with great success so far, using the antibodies and protocol obtained from Pharmingen. We've tried the stimulation a few different ways: 1) immediate staining of lymph node cells from primed (in our case infected) mice - no staining. 2) culture of LN cells from the mice for 5 hours with anti-CD3/anti-CD28 and monensin (the transport inhibitor you mentioned), then staining - some staining (this has also worked for human T cells). 3) culture of LN cells for 2-12 days with antigen-pulsed irradiated APC, monensin/anti-CD3/anti-CD28 for the final five hours - great staining. 4) as in (3), but LN cells from uninfected mice - little or no staining. 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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