Figure 3.

Determination of standard procedures for whole blood IFN-γ secretion assay. Diluted whole blood (1:5) was incubated with phorbol-12-myristate-13-acetate (PMA; 5, 10, 20, 50, 100 ng/ml), ionomycin (1 µM), and brefeldin A (1 µg/ml) for 2, 4, or 5 h, respectively. (A) The percentages of IFN-γ⁺ cells in CD4⁺, CD8⁺ T cells, and NK cells were evaluated by flow cytometry. Data are shown as mean ± SEM. (B) Diluted whole blood was stimulated with Leukocyte Activation Cocktail (BD GolgiPlug™, including 50 ng/ml PMA, 1 µM ionomycin, and 1 µg/ml brefeldin A) for 4 h. Representative flow cytometry gating strategy for identification of IFN-γ expression in CD4⁺, CD8⁺ T cells, and NK cells.