Anti tnf α pe mp6 xt22

The isolated lung, spleen and lymph node cells were stimulated in vitro in media containing 1 μg/mL anti-CD28 (37.51; BD Biosciences) and 1 μg/mL anti-CD49d (9C10; BD Biosciences) without antigen, or in the presence of 2 μg/mL H56 for 1 h, followed by incubation for 5 h at 37°C in the presence of 10 μg/mL brefeldin A (Sigma-Aldrich) and 0.7 μL/mL monensin/Golgi-stop (BD Biosciences). Following overnight storage at 4°C, the cells were washed with FACS buffer [PBS containing 0.1% (w/v) sodium azide and 1% (v/v) FCS] and stained for 30 min at 4°C for surface markers using anti-CD4-APC-eF780 (RM4-5; eBioscience, San Diego, CA, USA) and anti-CD44-APC (IM7; Biolegend) mAbs. For intracellular staining, cells were washed with FACS buffer, fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) and stained for 30 min at 4°C for intracellular cytokines using anti-IFN-γ-PE-Cy7 (XMG1.2; eBioscience), anti-TNF-α-PE (MP6-XT22; eBioscience), and anti-IL-17A-PerCP-Cy5.5 (eBio17B7; eBioscience). Cells were twice washed, resuspended in FACS buffer and analyzed using an LSRFortessa flow cytometer (BD Biosciences). Gates for the surface markers are based on fluorescence-minus-one controls. All flow cytometric analyses were performed using the FlowJo software v10 (Tree Star, Ashland, OR, USA).

Lymphocytes were isolated from spleens and lungs as described previously58 (link). Cultures were adjusted to 2 × 10⁵ cells/well for MSD/ELISA or 1–2 × 10⁶ cells/well in a total volume of 200 μl/well for flow cytometry and stimulated with antigens at a final concentration of 2 μg/ml. Concanavalin A was used at a concentration of 1 μg/ml as a positive control for cell viability. Culture supernatants were harvested after 72 h of incubation for the investigation of IL-17 ELISA. Lymphocytes for IC-FACS were stimulated at 37 °C in the presence of recombinant antigen (2 μg/ml) for 1 hour, and subsequently incubated for 6 hours after adding 10 μg/ml Brefeldin A (Sigma-Aldrich) and stained59 . The following antibodies were used for surface staining: anti-CD4-APC (clone RMA4-5, BD; 1:600), anti-CD44-FITC (clone IM7, eBioscience; 1:600). For intracellular staining, the following antibodies were applied in 1:200 dilutions: anti-IL-17A-PerCp-Cy5.5 (clone eBio17B7; eBiosciences), anti-IFN-γ-PE-Cy7 (clone XMG1.2; eBiosciences), anti-TNF-α-PE (MP6-XT22; eBiosciences), anti-IL-2-APC-Cy7 (clone JES6-5h4; BD). Responses were analyzed using a FACSCanto or LSRFortessa flow cytometer (BD) and FlowJo v.10.2 (Tree Star Inc.).