I am looking for the cytokines in cultured PBMCs that are stimulated in response to antigen. Mainly I am interested in cytokines secreted by T cells (th1/th2) and macrophages. I have already performed ELISA in cell supernatant and found stimulation of Th1 and Th17 cytokines (IFN-g, IL-2, IL-17). Now I want to do intracellular staining for these cytokines by using FACS Aria. As a beginner I only know that in FACS from FSC and SSC scatter plot, lymphocytes and monocytes can be separated. Further, whole T cell population can be selected by using CD3 marker, then Th and Tc by using CD4 and CD8 marker. For intracellular cytokine staining IFN-g, IL-4 and IL-17 can be used to see Th1,Th2 and Th17 response respectively. I want to know: 1. Markers that are needed to differentiate memory T cell from naive T-cells. 2. Markers and cytokines specific for macrophages 3. Along with IL-17 any other cytokine is required to see Th17 response (like IL-23).
Hello, here some comments/answers to your questions:
1. Several markers can be used to differentiate naive from the different memory T cell subsets, including CD27, CD62L, CD45RA, CD45RO, CD197 (CCR7),...I use CD45RA and CD197 antibodies to differentiate naive (positive for both markers), TCM (CD197+ CD45RAneg), TEM (negative for both markers) and Teff Diff (CD45RA+ CD197neg). Also, among naive cells, CD95+ cells are the newly described stem cell memory T cells (TSCM, see Roederer et al).
2. CD14 is a good marker for monocytes, CD16 can identify monocyte subsets. I am not familiar to cytokine production by monocytes but they are good producers of TNF alpha and IL-10 (after capture of apoptotic cells).
3. I suppose that IL-17 is sufficient to identify all TH17 cells (anyhow very few of them out there! ~1% of CD3 after PMA/Ionomycin stimulation in our hands). TNF-alpha co-production can be identified quite easily.
I suggest you to add a viability marker (amine-reactive dyes) in your assays for reducing your background due to dying cells.
Hope this helps.
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