Percp cy5.5 anti mhc 2

The phenotypes of DCs and BMDMs were determined by flow cytometry. For cell surface staining, single-cell suspensions were incubated for 15 min at 4 °C with PE-anti-CD11c (117308, 0.2 μg/mL), FITC-anti-CD80 (104706, 0.2 μg/mL), PE/Cy7-anti-CD86 (105014, 0.2 μg/mL), FITC-anti-MHC-I (114606, 0.2 μg/mL), PerCP/Cy5.5-anti-MHC-II (107626, 0.2 μg/mL), FITC-anti-F4/80 (123108, 0.2 μg/mL), PerCP/Cy5.5-anti-CD11b (101228, 0.3 μg/mL), PE-anti-CD206 (141706, 0.2 μg/mL), APC-anti-CD115 (135509, 0.4 μg/mL), FITC-anti-PDCA-1 (127007, 0.3 μg/mL), PerCP/Cy5.5-anti-CD44 (103032, 0.2 μg/mL), PE-anti-CD62L (104407, 0.2 μg/mL) (all purchased from Biolegend). Samples were washed and then analyzed by FACS versus flow cytometry (BD Biosciences).
For intracellular staining of cytokines, single-cell suspensions from DLN and spleen of the indicated EAE mice were stained with FITC-anti-CD4 (553047, BD Biosciences, 0.2 μg/mL) first, followed by staining with PE-anti-IFN-γ (554412, BD Biosciences, 0.3 μg/mL), PE-anti-IL17A (506904, Biolegend, 0.3 μg/mL) and PE-anti-IL4 (12-7041-82, eBioScience, 0.4 μg/mL) using Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s protocol. Intranuclear staining with PE-anti-Foxp3 (126404, Biolegend, 0.4 μg/mL) was performed using a Fixation/Permeabilization kit (eBioscience) according to the manufacturer’s protocol.

Apoptosis of BMDM were determined by using FITC Annexin V apoptosis detection kit with Propidium Iodide (PI) (BD biosciences), MHC-II expression of BMDM in response to rRv1768 stimulation was measured by PerCP/Cy5.5 anti-MHC-II (Biolegend, CA, USA). Cytokines production in culture supernatant of rRv1768 stimulated BMDM was determined using mouse Th1/Th2/Th17 cytometric-beads array kit (BD PharMingen, USA), according to the manufacturer's protocol. Intracellular IFN-γ expression of CD3⁺CD4⁺ T cells was determined by staining with APC-anti-CD3, FITC-anti-CD4, PE-anti-IFN-γ (Biolegend, CA, USA). All procedures were performed as we previously described (Tang et al., 2017 (link); Yuan et al., 2019 (link)) and analyzed on BD Accuri C6 Flow cytometer (BD Biosciences).