Cd86 24f

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).

Cell populations were stained with mAb against CD4 (W3/25), CD8 (Ox8), CD161 (10/78), TCR α/β chain (R73), CD25 (Ox39), CD86 (24F), CD11b/c (Ox-42) and CD172a (Ox41) (BioLegend, London, UK).