[Cytometry] Problem with intracellular interferon gamma staining

Stuart Berzins berzins at unimelb.edu.au
Fri Feb 19 22:23:25 EST 2010


Pre-incubating the anti-IFNg with murine IFN-g may be making your reagent
bind to both the receptor for IFN-g (via the bound cytokine) in addition to
the intracellular cytokine you're targeting. Hence, Its not surprising you
see more cells staining positive.

The reason you might be seeing IFN-g positive cells without restim is that
there is still an immune response going on in those mice. As the shams are
negative, presumably it is specific to the bacteria you're putting in.
Perhaps its taking a while to clear the infection and the residual antigen.
I'd try immunising with killed bacteria and see how that looks after 4
weeks. Or assay for the presence of the bacteria in those mice.

Regards,
Stuart


On 20/02/10 8:35 AM, "Reed, Douglas S" <dsreed at pitt.edu> wrote:

> I've got a problem with some intracellular cytokine staining and I'm hoping
> someone on this list might have the answer.
> 
> Here's the background. We're vaccinating mice with a live, attenuated strain
> of a bacterium.
> Four weeks later we harvest the spleens to look at cell-mediated immune
> responses. One of those assays is intracellular cytokine staining, looking for
> putatively 'polyfunctional' T cells but focusing particularly on interferon
> gamma. The cells are incubated overnight at 37oC; we add Brefeldin A at 19
> hours and recover the cells and stain them at 24 hours.
> 
> Here's the problem. In the animals that are vaccinated, we see a fairly good
> population of interferon gamma producing cells even without antigen
> restimulation in tissue culture (so in essence, what should be our negative
> control). We don't see these cells in the spleens of sham-vaccinated mice.
> 
> The concern was that these cells might be an artifact. We do use Fc Block, so
> that's not the answer. To try and address this, we tried pre-incubating the
> anti-interferon gamma antibody with recombinant murine interferon gamma and
> then staining the cells. The result we got back was an enhancement - more
> cells are staining positive than in the companion samples where we didn't
> preincubate with interferon gamma.
> 
> The antibody we're using is XMG1.2 from eBiosciences, which is listed as being
> neutralizing.
> 
> Thoughts?
> 
> Sincerely,
> Doug
> 
> Douglas S. Reed, Ph.D.
> Aerobiology Manager
> Center for Vaccine Research
> University of Pittsburgh
> (412) 648-9290
> dsreed at cvr.pitt.edu
> 
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Dr Stuart Berzins
NHMRC RD Wright Fellow
Department of Microbiology and Immunology,
The University of Melbourne,
Parkville 3010,
AUSTRALIA.
email: berzins at unimelb.edu.au
Ph (office): +61-3-8344-5706
Ph (lab): +61-3-8344-5704
Fax: +61-3-9347-1540
Mobile: 0427 849 123


 




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