Anti il 17a ebio17b7

At scarify, single-cell suspensions were isolated from dLNs, spleens, and CNS. Briefly, the brain and spinal cord were obtained, homogenized, and then incubated with collagenase D (2.5 mg/mL, Roche Diagnostics) and DNase I (1 mg/mL, MilliporeSigma) for 30 minutes. Mononuclear cells were enriched by gradient centrifugation at 670g for 30 minutes on a 37%/70% Percoll gradient without interruption. Before staining, cells were blocked with anti-CD16/CD32 antibodies. The following antibodies were used for flow cytometry: anti-CD3 (OKT3), anti-CD45 (30-F11), anti-MHCII (M5/114.15.2), anti-CD8 (53-6.7), anti-CD44 (IM7), anti-Foxp3(FJK-16S), anti–GM-CSF (MP1-22E9), anti-CD49d (R1-2), anti–IL-17A (eBio17B7), and anti–IFN-γ (XMG1.2) (eBioscience); anti-CD4 (GK1.5), anti-CD25 (PC61.5), anti-CD62L (MEL-14), anti–Ly-6G/Gr-1 (1A8-Ly6g),anti-F4/80 (BM8), anti-CCR2 (SA203G11), anti-CCR6 (29-2L17), and anti-CD29 (HMβ1-1) (BioLegend). In addition, anti–p-STAT3 (Y705) (catalog 557814) and p-STAT1 (Y701) (catalog 502069) antibodies were purchased from BD Biosciences. Phosphoflow cytometry analysis was performed using BD Phosflow buffers (554655 and 558050). Stained cells were analyzed by FACSCanto flow cytometer and were analyzed with FlowJo software (Tree Star).

The antibodies utilized in various experiments, along with their respective manufacturers, were as follows: anti-CD3 (145–2C11), anti-CD4 (RM4–5), anti-Gr1 (RB6–8C5), anti-CD11b (M1/70), anti- anti-IL-10 (JES5–16E3), IFNγ (XMG1.2), and IgG1 isotype controls were procured from BD Biosciences (San Diego, CA, USA). The following antibodies were obtained from eBioscience (San Diego, CA, USA): anti-CD45 (30-F11) and anti-IL-17A (eBIO17B7). Antibodies including anti-CD45 (30-F11), anti-CD19 (6D5), anti-CD11b (M1/70), anti-NK-1.1 (S17016D), anti-CXCR3 (CXCR3–173), and anti-CCR5 (HM-CCR5) were purchased from BioLegend (San Diego, CA, USA). The antibodies were conjugated with APC, FITC, PE, PerCP-Cy5.5, Alexa Fluor 647, or PE-Cy7 and appropriately paired.

Cytosolic Ca²⁺ concentrations were analyzed by flow cytometry. Cells were washed and stained with 3 μM Fluo-3AM (Biotinium, China) in Tyrode buffer (pH 7.4), cultured at 37°C for 30 min.
To determine intracellular change in ROS generation in CD4+ T cells, they were stained using a ROS assay kit (Beyotime). A total of 1 × 10⁵ cells were incubated with 10 μM carboxy-2’,7’-dichloro-dihydro-fluorescein diacetate probe to stain intracellular ROS in PBS for 30 min at 37 °C. Fluorescence was measured at 488 nm (excitation) and 525 nm (emission).
For cytokine staining, cells were re-stimulated with 1 μM ionomycin and 50 ng/mL PMA (phorbol myristate acetate) in the presence of 5 μM Brefeldin A (eBioscience) for 4 h. Cell surface staining was performed use CD4 Antibody (Invitrogen) in PBS, followed by permeabilization with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intercellular staining in permeabilization buffer (eBioscience). The following antibodies were used: anti-IL-17A (eBio17B7; eBioscience) antibodies and anti-Foxp3 (FJK-16s; Invitrogen) antibody.

For intracellular cytokine staining, cells were stimulated for 5 h with a cell stimulation cocktail (eBioscience), followed by surface staining with CD4 for 30 min at 4 °C. To stain intracellular antigens and antibodies, cells were permeabilized with IC Fixation Buffer and Permeabilization Buffer (eBioscience) following the manufacturer’s instruction and stained with anti-IFN-γ (DB-1; Invitrogen), anti-IL-17A (eBio17B7; eBioscience), anti-IL-4 (OX81; eBioscience), anti-IgG (Poly4054; Biolegend) or anti-Ig light chain κ (MRG81; Biolegend), respectively. Stained cells were analyzed with BD AriaIIflow cytometer and Flowjo software (TreeStar Inc., Ashland, OR, USA).
For regulatory T (Treg) cells staining, cells were stained with surface marker CD4 (OX35; eBioscience) and CD25 (OX39; Invitrogen) before being fixed and permeabilized with Perm/Fix solution (eBioscience). Finally, the antibody for Foxp3 (FJK-16S; eBioscience) was added for 30 min in the dark. Stained cells were analyzed with a flow cytometer and Flowjo software.