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.