Representative flow cytometry protocol to determine the frequencies of CD8+T-lymphocyte subsets and apoptosis. Peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients were stained ex vivo and after antigenic-stimulated cultures with CD3-FITC, CD8-APC, CD45RA-ECD, CD27-PE-Cy7 and 7-AAD. The lymphocytes were gated on forward (FSC) vs side (SSC) scatter dot-plot (A), backgated from CD3+ histogram and doublets were excluded by a density plot of FSC Area vs FSC Width (B). CD27 vs CD45RA dot plot (D) gated on CD3+CD8+ region (C) was used to define the frequencies of effector and naïve CD8+ T lymphocytes. Frequency of apoptotic cells (7AADlow) was determined by FSC vs 7AAD dot-plot gated on CD3+CD8+ cells (total CD8+ T lymphocytes) (E) and on CD45RA+CD27neg cells (effector CD8+ T lymphocytes) (F).