Anti ifn γ xmg1

After homogenizing spleens, cells were treated with erythrocyte lysis buffer, meshed through a 35-μm cell strainer, washed with PBS, and analyzed by FACS.
DCs were phenotyped by using fluorochrome-labeled mAbs directed against CD11c (N418; BioLegend), CD80 (16-10A1; BD Pharmingen, Heidelberg, Germany), CD86 (PO3; Thermo Fisher Scientific), PD-1 (J43, Thermo Fisher Scientific), CTLA-4 (UC10-4B9; BioLegend), CD83 (Michel-19; BioLegend), CD40 (3/23; BioLegend), MHCII (2G9; BD Pharmingen) and PD-L1 (MIH6; AbD Serotec, Puchheim, Germany). Dead cells were excluded using the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Following mAb labeling for 30 min at 4 °C and final washing, cells were analyzed on an LSR II flow cytometer.
To measure intracellular cytokines, cells were stimulated with PMA and ionomycin (1 μg/ml each; Sigma-Aldrich, Taufkirchen, Germany) for 4 h in the presence of 3 μg/ml Brefeldin A (Thermo Fisher Scientific), labeled for CD11c, subjected to fixation and permeabilization and subsequently stained with mAbs against IL-10 (JES5-16E3; BD Pharmingen) and IL-12 (C15.6; BD Pharmingen). IFN-γ in T cells was measured by using fluorochrome-labeled anti-IFN-γ (XMG-1.2, BioLegend) and counterstaining with anti-CD4 (RM4-5, eBioscience, Frankfurt, Germany).

Anti-CD4 (RM4-5), anti-CD8a (53–6.7), anti-CD44 (IM-7), anti-CD25 (PC61), anti-Cd11b (M1/70), anti-TER-119 (TER-119), anti-IgM (R6-60.2), anti-TCRβ (H57-597), anti-CD62L (MEL-14), anti-CD69 (H1.2F3) anti-IL-6 (MP5-20F3) and TNF-α (MP6-XT22) were purchased from BD Pharmingen. Anti-B220 (RA3-6B2) and anti-CD3 (145-2C11), anti-IFN-γ (XMG1.2) were purchased from BioLegend. Anti-CD43 (GL3) was purchased from eBioscience (eBioR2/60). PMA and ionomycin were purchased from Merck. Cycloheximide was purchased from Sigma. MG-132 was obtained from Merck. Taq Plus Master Mix for genotyping was from Vazyme Biotech. Chloroquine was obtained from MCE. Collagenase IV was obtained from Sigma. DNase I was obtained from Shanghai Sanjie.

Single-cell suspensions were incubated with the Fc blocking antibody (2.4G2) and with fixable blue dead cell stain kit (Invitrogen). To stain surface molecules the following antibodies were used: anti-CD3 (145-2C11), anti-CD11b (M1/70), anti-CD19 (1D3), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD49a (Ha31/8), anti-Ly6C (AL-21), anti-Ly6C/Ly6G (RB6-8C5), anti-Ly49H (3D10), anti-MHCII (M5/114.15.2), anti-NK1.1 (PK136), and anti-TCRβ (H57-597) from BD Biosciences; anti-NKp46 (29A1.4), anti-CD49b (DX5 or HMa2), anti-F4/80 (BM8), and anti-Ly49H (3D10) from eBioscience; and anti-CD11c (N418) and anti-Ly6G (1A8) from Biolegend. For intracellular staining, the cells were fixed and permeabilized with an intracellular staining kit (eBioscience), and the following antibodies were used: anti-IFN-γ (XMG1.2) from Biolegend, anti-Ki67 (B56) from BD Biosciences, and anti-GzB (GB12) from Life Technologies. Analysis was performed with FlowJo Software.

Cells were stimulated for 4 h with PMA (phorbol 12-myristate13-acetate; 50 ng/mL), ionomycin (1.0 µg/mL), and a protein-transport inhibitor containing monensin. PMA and ionomycin stimulate the immune cells in a non-antigen specific manner, and monensin is used to trap the cytokine within the cytosol. After stimulation, surface markers were stained for 15–20 min at room temperature in PBS with 1% FBS. Cells were then fixed in Cytofix and permeabilized with Perm/Wash Buffer using the BD Fixation Permeabilization solution kit and stained with anti-IL-17A (TC11-18H10.1, Biolegend, San Diego, CA, USA); anti-IFN-γ (XMG1.2, Biolegend); and anti-IL-4 (11B11, Biolegend) antibodies diluted in Perm/Wash buffer. Permeabilization was undertaken in order to make the intracellular cytokines accessible to the FACS antibodies. All antibodies were used in 1:500 dilutions. Flow cytometry was undertaken using BD FACS Symphony, and the data were analyzed with FlowJo software version 10.9.0.