Anti tnfr2 apc

Flow cytometry was performed using a 10-color Navios Flow cytometer (Beckman Coulter, California, United States), which is equipped with blue (488 nm), red (638 nm), and violet (405 nm) lasers. For surface staining, the following antibodies were used: anti-CD3-ECD (UCHT1), anti-CD45RA-ECD (2H4LDH11LDB9), anti-CD45-KO (J33), anti-CD4-PE-Cy5.5 (13B8.2), and anti-CD8-APC-AF700 (B9.11) (all from Beckman-Coulter); anti-TNFR1-AF488 (16803, R&D); and anti-TNFR2-APC (22235, R&D). For intracellular staining, the following antibodies were used: anti-IFNγ-PE-Cy7 (4S.B3) and anti-IL-17A-AF-660 (eBio64DEC17) (eBioscience, California, United States). Unstained (Fluorescence Minus One, FMO) samples were also measured to help set the gates during data analysis. To evaluate cytokine production, we challenged the cultured Treg subsets for another 4 h with PMA (12.5 ng/ml), ionomycin (500 ng/ml), and Brefeldin A (5 μg/ml) (Sigma-Aldrich, Missouri, United States) before performing the FACS staining process. Briefly, cells were stained with the fixable viability dye-eFluo 780 (FVD, eBioscience) for 30 min at 4°C, following with surface mAb staining, cell fixation, and permeabilization by using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience) and intracellular mAb staining. For flow cytometry data analysis, Kaluza1.5 software (Beckman Coulter) was used.

Phenotypic characteristics of immune cells were assessed by flow cytometry (FACSVerse cytometer; BD, USA) using monoclonal antibodies: anti-CD25-FITC, anti-CD45R0-FITC, anti-CD19-PE-Cy7, anti-CD4-PE-Cy7, anti-CD3-PE-Cy7, anti-CD8-PE-Cy7, anti-CD127-PE-Cy7, anti-CD14-PerCP, and anti-CD45RA-Pacific Blue (Biolegend, USA); and anti-TNFR1-PE, anti-TNFR2-PE, anti-TNFR1-APC, and anti-TNFR2-APC (R&D Systems, USA). Data processing and calculation of fluorescence intensity parameters were performed in the FacsDiva software (BD, USA). To calculate the number of receptor molecules per cell, the BD QuantiBRITE PE Kit (BD Biosciences, USA) was used according to the method described earlier [22 (link)].