Fig. 2.

Gating strategy for defining antigen-specific T-cell functions using flow cytometry. This figure summarizes procedures for demarcating antigen-specific T-cells using FlowJo software, FlowJo, LLC. (i) First, lymphocytes were identified; (ii) plotted on forward scatter height (FSC-H) against forward scatter area (FSC-A) to select single cells, (iii) subjected to CD14, CD19, and Aqua antibody exclusion to remove B-cells, monocytes, and dead cells, respectively, (iv) before gating out CD3+ lymphocytes, A. Double positive T cells (CD3+CD4+CD8+) were excluded, while CD4+ (CD3+CD8−CD4+) and CD8+ (CD3+CD4−CD8+) T cells were selected for subsequent evaluations, B. Secretion of IFN-γ, IL-2, TNF-α, and Perforin was then quantified using cytokine-specific plots derived from FlowJo software, C. Finally, the co-expressed T cell functions were further distinguished into singly expressed, concurrently expressed, and non-functional subsets using FlowJo generated Boolean gating.