[Cytometry] Problem with intracellular interferon gamma staining

Ruud Hulspas rhulspas at cytonomest.com
Sat Feb 20 15:53:05 EST 2010


So, there are two negative controls here. I think your negative control 
for the staining is the sample from the sham-vaccinated mice and that 
the 'problem' may be in the method that prepares the other negative 
control. I assume that you'll find interferon gamma positive cells right 
after stimulation. It'll take a while before those first responders 
disappear in tissue culture. You may want to try to wait longer before 
re-stimulating.

Ruud

On 2/19/2010 4:35 PM, Reed, Douglas S 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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>
>    

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*Dr. Ruud Hulspas, Ph.D.*
Scientific Director

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RHulspas at cytonomest.com <mailto:RHulspas at cytonomest.com>



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