Anti mouse cd16 32 2.4g2

EAE mice were sacrificed on day 20, and mononuclear cells from spleens were separated by using density gradient centrifugation. The cells were co-cultured in 24-well plate (1×10⁶ cells/well) with MOG_35–55 or MOG_40–54 peptides (25 μg/mL) for 16 h at 37°C under 5% CO₂ and humidified conditions, followed by the addition of protein transport inhibitor Brefeldin A (Multi Sciences, Shanghai, China) for another 5 h. Cells were harvested and blocked with anti-mouse CD16/32 (2.4G2; eBiosciences) for 20 min and then stained with FITC-anti-mouse CD4 and APC-anti-mouse CD8a (eBiosciences) for 30 min at 4°C. After that, the cells were incubated with PE-anti-mouse interferon (IFN)-γ (XMG1.2) or PE-anti-mouse IL-17A (eBio17B7) (eBiosciences) for 30 min at 4°C after fixation/permeabilization and analyzed by flow cytometry. The gating strategy for each figure of flow cytometry was described in Supplementary materials.

Enucleated eyes were dissected in 100 μL cold Dulbecco's modified Eagle's medium. After incision at the limbus with a 29-gauge needle, a circumferential cut around the eye following the limbus was made. Iris was dissected, releasing anterior chamber infiltrating cells into the dissection media. The retina and vitreous were then removed carefully from the eye cup, leaving the sclera/retinal pigment epithelium/choroid intact. The dissection media containing anterior fluid, vitreous, and retina were then pipetted into a 1.5-mL Eppendorf tube and mechanically disrupted by rapping 10 times for a single-cell suspension, followed by centrifugation through a 70-μm cell strainer. The resulting single-cell suspensions were blocked with anti-mouse CD16/32 (2.4G2; eBioscience, Waltham, MA) for 5 minutes prior to surface staining with the following antibodies: αGr1-APC, αLy6C-PE, αCD11b-PerCP, αCD45-FITC, APC-efluor780 live dead dye. Absolute cell numbers were obtained as previously detailed^{25 (link)} using a BD LSR Fortessa-x20 with FACSDiva software (version 8.1; BD Cytometry Systems, San Jose, CA). Single-stained beads (OneComp eBeads; eBioscience) of all fluorophores were used to generate compensation matrices, and fluorescence-minus-one controls were used for positive gating; data were analyzed using FlowJo software (Treestar).