Figure 10.

Gating strategy for flow cytometric data analysis and calculation of the absolute cell counts of lymphocyte subsets. Lymphocytes were identified based on forward and side scatter (FSC/SSC) properties, and then gated for expression of CD4 surface receptor. CD4⁺ T cells were analyzed for expression/co-expression of CD25 and Foxp3. On this basis, Foxp3⁺CD25⁺CD4⁺ and Foxp3⁻CD25⁺CD4⁺ T cells, i.e., regulatory (Treg) and activated effector (aTeff) T cells, respectively, were distinguished. Subsequently, IL-10- and TGF-β-producing cells as well as 5-bromo-2-deoxyuridine(BrdU)-incorporating cells were identified within these cell subsets. Moreover, CD39-expressing cells were identified within Treg cell subset. In addition, type 1 regulatory T (Tr1) cells were detected on the basis of CD49b and CD223 co-expression within CD4⁺ T cell subset. Absolute cell counts of lymphocyte subsets (i.e., number of cells from particular subpopulations per sample well) were calculated using the dual platform method, as shown above.