Working with murine spleenocytes. I stimulate 1 million cells with 50ng/ml PMA, 2ug/ml Ionomycin, 10ug/ml Briefeldin A for 4 hours at 37degC 5%CO2, followed by intracellular staining for cytokines, primarily IFNy. Unstimulated cells show more production of cytokines than stimulated cells. Is it better to add BFA along with PMA/Ionomycin or should I incubate cells with PMA/Ionomycin for 1-2 hours before adding BFA? When exactly should BFA be added?
Any other tips?
When I stimulated splenocytes or T-cells with PMA/Ionomycin (PMA - 50 ng/ml, Ionomycin - 500 ng/ml), I always gave them 1 hour for activation and only then added BfA for another 4-5 hours. CD4 T-cells show pretty good stimulation, CD8 T-cells show very moderate cytokine response. Although in most of the protocols the guys don`t say about this first incubation.
Actually, I never liked this method as in my hands CD8 T-cells rarely showed strong cytokine response :)
BTW, are you sure that your cells or antibody staining are good? There might be also non-specific antibody staining.
Thank you for your response!
The antibody has shown pretty good staining in other tissues, even without stimulation. I also set up FMO controls to rule that out, but thanks for the tips, I'll test out other colors for the same antibody.
It may also may depend on the cytokine you test and the cell type.
In my hands, CD8 T-cells from the splenocytes of naive mice rarely give good IFNg response, but peptide-stimulated splenocytes from virus-infected mice gave very strong responce. And CD4 T-cells from naive mice showed relatively good expression of inflammatory cytokines (IFNg, IL2, TNFa), but IL10 and TGFb - almost nothing.
And if you work with myeloid cells, they may be much more tricky. In my hands, after PMA they sticked to the plastic very firmly.
There is a pretty good manual about this method on the website of Biolegend.
Oh! I am trying to detect IFNy, which should show up in spleenocytes without many problems. But thank you for the information, it is really helpful!
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