Anti cd8 apc af700 b9.11

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.

Spleen and mesenteric lymph nodes (LN) were harvested and cells were obtained by mashing over a 40 µm filter. Single cell suspensions of spleen and LN were phenotypically analyzed using a Navios multi-color flow cytometer (Beckman-Coulter, Mijdrecht, the Netherlands). Human leukocytes were identified using an antibody directed against the common human surface leukocyte marker CD45 (anti-human-CD45-KO (J33, Beckman-Coulter). For cell-surface staining of human CD4 and CD8 T cells, the following antibodies were used: anti-CD4-PC5.5 (13B8.2, Beckman Coulter), and anti-CD8-APC-AF700 (B9.11, Beckman-Coulter). For intracellular staining with anti-FOXP3-e450 (PCH101, eBioscience), cells were fixed and permeabilized by fix-perm treatment (eBioscience) according to manufacturers instruction. Data were analyzed using Kaluza software version 1.5a (Beckman-Coulter) and gates were set based on single staining and FMOs (Supplemental Fig. 1)