Hi, thanks to all that wrote back about my question regarding detection of intracellular IFN-g by flow cytometry, the information was helpful. It was my first time writing in, and I think I should have given more information. - I use a PE-labelled anti-IFN-g antibody from R&D and the cytofix/cytoperm kit from PharMingen. - I can detect IFN-g really well from T cells and NK cells if I use PMA/Ionomycin - When I use my protein of interest as a stimulus for PBMC, I can't detect any IFN-g inside of T cells or NK cells - However, I can detect up-regulation of IFN-g mRNA (by PCR) and secreted protein (by ELISA), so I know it is being produced. - I know that others in the literature use anti-CD28 as a co-stimulus (even while using strong superantigens like SEB) to help detect IFN-g by T cells because detection levels are quite low (about 5% in T cells if you use SEB and anti-CD28 as a stimulus) - The question that I want to answer is whether T cells or NK cells, or both produce IFN-g What I haven't found, after searching through the literature, is if there are any other techniques that people use to help detect IFN-g (when using stimulus that is much weaker than PMA/Ionomycin). Any more info would be greatly appreciated. Thanks, Slava Epelman sepelman@acs.ucalgary.ca Dept of Microbiology and Infectious Diseases University of Calgary
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