Apc conjugated il 10

Using flow cytometry, circulating Bregs were detected using FITC-conjugated-CD38, PE-conjugated-CD24 (Bioscience, USA), and PerCP-conjugated CD19 (BD Bioscience, USA). Briefly, 100 µl of blood sample was incubated with 10 µL of CD24, CD38 and CD19 for 20 min at 4 °C in the dark. Following incubation, RBCs were lysed and washed. Cells were fixed and permeabilized then stained with APC-conjugated IL-10 (BD Bioscience, San Jose, CA, USA) and analysis by FACS Calibur flow cytometry with CellQuest software (Becton Dickinson Biosciences, San Jose, CA, USA). An isotype-matched negative control was used for each sample. Forward and side scatter histograms were used to define the lymphocytes population. CD19⁺ IL-10⁺ B cells were gated, then the expression of CD38 and CD24 on the CD19⁺B cells were detected. Bregs were identified as CD19⁺ IL-10⁺CD24^+hiCD38^+hi cells. CD19⁺ B cells were selected based on the use of the isotype-matched negative control. However, for proper gating of the IL-10⁺ and CD24^+hiCD38^+hi cells, fluorescence minus one controls were employed, shown in light blue and red colors in the dot blots for the gated cells and controls, respectively (Figure 1A).

PC5.5-conjugated CD4 (BD Pharmingen™; cat: 560650), PC7-conjugated CD25 (BD Pharmingen™; cat: 557741), PB450-conjugated ICOS (BD Pharmingen™; cat: 562901) or PB450-conjugated GITR (BD Pharmingen™; cat: 747658) were used for surface staining. Consequently, the cells were fixed and permeabilized. PE-conjugated Foxp3 (BD Pharmingen™; cat: 560650), APC-conjugated CTLA4 (BD Pharmingen™; cat: 555855) or APC-conjugated Helios (BD Pharmingen™; cat: 560046) were used for intracellular staining.
To analyze the production of cytokines, PBMCs (2 × 10⁶ cells/ml) were incubated in RPMI 1640 culture medium containing 10% fetal bovine serum (Sigma; cat no. 030M3399) with 50 ng/ml phorbol myristate acetate (PMA) (Sigma; cat: P1585-1MG) and 1 µg/ml ionomycin (Sigma; cat no. I0634-1MG) in the presence of 0.7 µl/mL GolgiStop (BD Biosciences; cat: 554724) and 1 µl/mL Golgi Plug (BD Biosciences; cat: 555029) for 6 h according to the manufacturer’s instructions. Surface staining for CD4 and CD25 and intracellular staining for Foxp3, PB450-conjugated IL-17A (BD Pharmingen™; cat: 562933), FITC-conjugated IFN-γ (BD Pharmingen™; cat: 554700), or APC–conjugated IL-10 (BD Pharmingen™; cat: 554707) were the same as described above.