Pe cy7 conjugated anti cd19

A CNS and spleen leukocyte suspension was obtained as described previously (Carrillo‐Salinas et al., 2017 (link)). Isolated cells were incubated with anti‐CD16/CD32 (Affymetrix Inc.) for FcR blockade and labeled with anti‐mouse antibodies: PE‐conjugated anti‐CD44 (2.4 μg/ml), PE‐conjugated anti‐CD274 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD4 (1.2 μg/ml), PECy7‐conjugated anti‐Ly6c (1.2 μg/ml), APC‐Cy7‐conjugated anti‐CD11b (1.2 μg/ml), APC‐conjugated anti‐CD62L (2.4 μg/ml), APC‐conjugated anti‐CD25 (2.5 μg/ml), and APC‐conjugated anti‐CD1d (2.4 μg/ml; all from eBioscience); APC‐conjugated anti‐P2yR12 (2.5 μg/ml) and APC‐Cy7‐conjugated anti‐CD3 (1.2 μg/ml) (both from Biolegend); PE‐conjugated anti‐CD5 (2.2 μg/ml), PerCP‐Cy5.5‐conjugated anti‐B220 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD45 (1.2 μg/ml), PECy7‐conjugated anti‐CD8 (1.2 μg/ml) and PECy7‐conjugated anti‐CD19 (2.4 μg/ml; all from BD Pharmingen). The cells were fixed for 30 min with fixation buffer (Affymetrix Inc.). For FoxP3 detection, the cells were suspended in Fixation/Permeabilization buffer for 30 mins prior to staining with an anti‐FoxP3 antibody (3 μg/ml; BD Pharmingen). At least 50,000 events were registered in each experiment on a FACSAria flow cytometer (BD Biosciences), excluding duplets from the analysis. The data were analyzed using FACSDiva analysis software (BD Biosciences).

A CNS or spleen leukocyte suspension was obtained as described previously (30 (link)). Freshly isolated cells (10⁶) were incubated with a Fc block (anti-mouse CD16/CD32: eBioscience, San Diego, CA, USA) and labeled with anti-mouse antibodies: PE anti-CD25; PE anti-CD3; PerCP-Cy5.5-conjugated anti-CD45; PerCP-Cy5.5-conjugated anti-B220; PE-Cy7-conjugated anti-CD39; PE-Cy7-conjugated anti-CD8a; PE-Cy7-conjugated anti-CD19; APC anti-CD3; APC-Cy7-conjugated anti-CD4; APC-Cy7-conjugated anti-CD11b (all from BD Pharmingen, San Diego, CA, USA); APC anti-CD3; and PE-Cy7-conjugated anti-CD39 (both from eBioscience, San Diego, CA, USA). For Foxp3 detection the cells were suspended in fixation/permeabilization buffer for 30 min and stained with a PE anti-Foxp3 antibody (BD Pharmingen, San Diego, CA, USA). Cell viability was assessed with the LIVE/DEAD Fixable Green Dead Cell Stain Kit or LIVE/DEAD Fixable Red Dead Cell Stain Kit (Life Technologies Corporation, OR, USA). At least 10,000 events were acquired in each experiment on a FACSaria flow cytometer (BD Biosciences, San Diego, CA, USA) and the data were analyzed using FlowJo software (v.10; Tree Star, Ashland, OR, USA).

PBMCs (1 × 10⁶ cells/ml) were stimulated for 48 h with rIL-35 (100 ng/ml; Sino Biological, Inc.). The cultured cells in duplicate were stained with surface FITC-conjugated anti-CD3 (cat. no. 349201; BD Biosciences) and PE–cy7–conjugated anti-CD19 (cat. no. 341113; BD Biosciences) at room temperature for 15 min in the dark. The cells were subsequently fixed with 50 µl BD FACS™ permeabilizing solution A (cat. no. 347692; BD Biosciences) at room temperature for 5 min in the dark according to the manufacturer’s instructions and then permeabilized with BD FACS™ permeabilizing solution B (cat. no. 347692; BD Biosciences) and incubated with intracellular APC-conjugated anti-IL-10 (cat. no. 554707; BD Pharmingen; BD Biosciences), PE-conjugated anti-phospho-STAT1 (anti-p-STAT) (cat. no. 8062; Cell Signaling Technology, Inc.), PE-conjugated anti-p-STAT3 (cat. no. 87544; Cell Signaling Technology, Inc.), PE-conjugated anti-p-STAT4 (cat. no. 13223; Cell Signaling Technology, Inc.), or isotype-matched controls at room temperature for 30 min in the dark. After washing with PBS, the cells were subjected to flow cytometry analysis using a FACS Canto II instrument (BD Biosciences), and data were analyzed by FACS Diva software (version 6.1.3; BD Biosciences).