Cd4 ox35

For phenotype characterization of white blood cells, a multi-parameter analysis was carried out by flow cytometry (Navios, Beckman Coulter®). Whole blood was collected in tube field with cellular antigen stabilization solution (Transfix, Cliniscience) and kept at 4 °C up to 14 days. Red blood cells were lysed with Macs lysing solution. To minimize non-specific antibody binding, after centrifugation, cells were incubated in a buffer containing PBS, 2% human albumin (LFB), and 2 μg/mL polyvalent human immunoglobulin (R&D Systems) before staining. Cells were then incubated with fluorescent monoclonal antibodies: CD8a (OX8, eBioscience), CD4 (OX35, eBioscience), CD3 (1F4, BioRad), CD80 (3H5, BioRad), CD279 or PD-1 (KLH, Bioss), CD274 or PD-L1 (KLH, Bioss), CD11b/c (REA, Miltenyi), CD28 (REA, Miltenyi), CD45 (REA, Miltenyi), CD86 (REA, Miltenyi), and MHC2 (REA, Miltenyi), at a saturating concentration for 20 min at + 4 °C. Isotype antibodies, non-stain cells, and fluorescence minus one were used as controls. The FlowJo software was used to set up gating and analyze positivity frequencies of the makers of interest.

On day 50 (peak of the disease) after induction of EAMG, rats were euthanized under deepening anesthesia. Spleens and inguinal lymph nodes were removed and the weights were measured. Then the spleens and inguinal lymph nodes were minced through a 70 μm cell strainer to prepare single-cell suspensions of mononuclear cells (MNCs) under aseptic conditions. Red blood cells in spleen MNCs were lysed with a lysis buffer (Biolegend) for 5 min. All specimens were coded to facilitate blind testing. The spleen or inguinal lymph nodes MNCs suspension with a final volume of 100 μl were labeled with corresponding monoclonal antibodies and incubated for 30 min at 4 °C in the dark. The following antibodies were used in the assay: CD3 (1F4; Biolegend), CD4 (OX35; eBioscience), B220 (HIS24; eBioscience), CD20 (SP32; Abcam), CD161 (10/78; BD Pharmingen), MHC II (HIS19; Biolegend), CD80 (3H5; Biolegend), CD86 (24F; Biolegend), CD40 (HM40-3; Biolegend), CD27 (LG.7F9; eBioscience), ICOS (C398.4A; Biolegend), CXCR5 (ERP8837; Abcam). Unconjugated primary antibodies were detected with Alexa Fluro 488-conjugated anti-rabbit IgG (Abcam). Samples were analyzed using an Aria II flow cytometer (BD).