I am trying to do IFN-γ intracellular staining of mouse spleen cells and PBMC. I will stimulate cells with PMA and ionomycin first. I want to know what percentages of CD4+ T cells or CD8+ T cells are IFN-γ positive in spleen and PBMC of wild type B6 mice?
Thanks in advance!
Hi, I am not an expert for spleen, and only a partial one for PBMC, but you could have some information about human PBMC in my old paper dated 1995. Distinct pattern of IL-2 and IFN-gamma gene expression in CD4 and CD8 T cells: cytofluorometric analysis at a single cell level using non-radioactive probes. Cellular and molecular biology 12/1995; 41(7):945-57. best regards, Pierre-Yves Morvan
It depends on the duration of stimulation and if you use cytokine secretion blocking agents (BREFALDIN A /momensin) or not. Splenocytes stimulated for 24hrs with last 8 hours blocking by Brefaldin A gives around 20 percent of IFNgamma positive CD4 cells.
For PBLwhich was cultured overnight with or without CD3/CD28 then stimulated by PMA/Ionomycin/BFA for 3 hr, I saw about 6% in CD4 and 20% in CD8 without CD3/CD28 sti, and 10% in CD4 and 25% in CD8 with CD3/CD28 sti
I just gave you positive IFN-gamma in human PBL cells. As for mouse speen cells, we see about 5 - 6 % in CD3+4+ or CD3+8+ without ConA sti or 6 % in CD3+4+ and 16% in CD3+8+ with ConA sti overnight
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