Percp cy5.5 conjugated anti cd45

Aortas were dissected, minced using scissors and enzymatically digested with 200 U/mL Liberase (Roche) and 40 U/mL DNase I (Sigma Aldrich) in HBSS plus 5% FCS for 1 h at 37°C. Cells were filtered through a 40 μm nylon strainer, washed with HBSS plus 5% FCS, collected by centrifugation at 400 g for 5 min at 4°C and then suspended in FACS buffer (PBS plus 0.2% FCS plus 1 mM EDTA). Murine Fc receptors were blocked using anti-CD16/32 antibodies (BioLegend) for 10 min on ice. Violet 510 Viability Dye (Cell Signaling Technology) was used to discriminate between live and dead cells. The cells were stained with the following antibodies for 30 min at 4°C: PerCP-Cy5.5-conjugated anti-CD45, APC-Cy7-conjugated anti-CD11b, FITC-conjugated anti-Ly6G, PE-Cy7-conjugated anti-F4/80, and APC-conjugated anti-Ly6C (all purchased from BioLegend). Flow cytometric analysis was performed using FACSCanto II flow cytometer (BD Biosciences) and DIVA Software (BD Biosciences).

Single‐cell suspensions from the LN, spleen, and the CNS of mice were stained with extracellular fluorochrome‐conjugated CD antibodies (Biolegend), along with APC‐conjugated MOG_38‐49 I‐A^b tetramer (NIH tetramer core, peptide sequence: GWYRSPFSRVVH) and Brilliant Violet 421‐conjugated human CLIP_87‐101 I‐A^b tetramer as a control (peptide sequence: PVSKMRMATPLLMQA). Then, cells were fixed and permeabilized using the kit (eBioscience, Cat# 00‐5523‐00) for intracellular staining of PE‐conjugated anti‐FoxP3 (eBioscience Cat# 12‐5773‐82) per the instruction. For intracellular staining of CNS isolated immune cells, cells were stimulated with PMA (5 ng/ml), ionomycin (500 ng/ml), and protein transport inhibitor (0.7 µl/ml, BD Biosciences. Cat# 51‐2092KZ) for 5hrs, followed by extracellular marker and intracellular cytokine antibody staining. Data were analyzed using FlowJo™ v10 software. Mean fluorescence intensity (MFI) was defined as the “medians” of fluorescence intensities of the conjugated fluorochromes of the antibodies.
Cells isolated from the CP were incubated with PMA, ionomycin, and protein transport inhibitor as above for 5 h, followed by extracellular staining with PerCP/Cy5.5 conjugated anti‐CD45 (Biolegend, Cat# 103132) and intracellular staining with APC‐conjugated anti‐IFN‐γ (Biolegend, Cat# 505810).