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. 2018 Dec 7;9:2891. doi: 10.3389/fimmu.2018.02891

Figure 4.

Figure 4

Scheme of Flow-based quantification of various intracellular cytokines production, and of different types of CD4+ cell, and different cytokine producing CD8+ cells in infected mice after in vitro stimulation with PMA+IMB. After cell sorting for CD45+ cells (top left), cells were stimulated and then marked with antibodies against mouse CD4 (FITC), and CD8 (AF 647) cells (top center). Cells were then fixed, permeabilized, and stained for intracellular cytokines: IFN-γ-PB, TNF-α-APC.Cy7, IL-21-e-Fluor 711, IL-10-PerCP/Cy5.5, and IL-4-BV605 (2nd row). Then CD4+ cells producing different cytokines were quantified (results presented in Figure 5A). In CD4+ subpopulation, cells that were positive for IFN-γ, but negative for other four cytokines were identified as Th1 cells (bottom left). CD4+ cells that had IL-10 but no other cytokines were defined as Tregs, whereas cells producing both IL-4 and IL-10 cytokines, but not IL-21, IFN-γ, and TNF-α were identified as Th2 (bottom center two boxes). CD4+ cells that produced only IL-21 were identified as Th17 (bottom right) and results are presented in Figure 5B). CD8 cells producing different cytokines are presented in Figure 6.