Analysis of co-expression of CD26 with each of the cytokines or markers of different subpopulations of lymphocytes by flow cytometry
All the cell labelling with immune fluorescence-conjugated antibodies was performed in 1% (w/v) BSA/PBS at 4°C for 1 h in the dark. For the determination of co-expression of CD26 with cell surface markers of lymphocyte subpopulations, lymphocytes were incubated with both FITC-conjugated anti-human CD26 and PE-conjugated corresponding antibody simultaneously. For determination of the co-expression of CD26 with intracellular cytokines, after incubation with FITC-conjugated-anti-human CD26, the cells were washed and fixed with 4% formaldehyde for 5 min, washed again and subsequently permeabilized with 0.1% Triton X-100 in PBS for 10 min. After further wash steps after permeabilization, the cells were then incubated with PE-conjugated corresponding antibody. Fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated antibodies (direct against CD26, CD4, CD8, CD69, CD25, CD71, IL-2, IFN-γ, IL-4, IL-13, IL-6, IL-17, IL-22, IL-23R), as well as allophycocyanin (APC)-conjugated anti-FoxP3 antibodies were obtained from ImmunoTools (Friesoythe; Germany). Allophycocyanin (APC)-conjugated anti-CD161 and Per-conjugated anti-CD196 antibodies were provided by MACS Miltenyi Biotec (Bergisch Gladbach, Germany). After the immunofluorescent cells were resuspended in FACS buffer and measured by flow cytometry, the WinMDI 2.9 software was used to analyze the percentages of different lymphocyte subpopulations or cytokine secreting cells.