Facsdiva v8

For immunofluorescence staining, morphology of cells was determined via FSC-A and SSC-A, doublets excluded via FSC-A and FSC-H and dead cells excluded with Fixable-Yellow live/dead stain (Invitrogen, Carlsbad, CA, USA). Human and mouse MR1 tetramers were produced^{13 (link), 53 (link)}. Biotinylated MR1-5-OP-RU and control MR1-Ac-6FP monomers were tetramerized with streptavidin conjugated to PE (BD Pharmigen; San Jose, CA, USA). For intracellular detection of IFN-γ and TNF, cells were cultured for 4 h with 5 ng/mL PMA and 1 µg/mL Ionomycin in the presence of Golgi Plug and Golgi Stop (BD Pharmingen; San Jose, CA, USA). Data were collected using a BD FACSymphony and analyzed using FacsDiva v8 and FlowJo v10.2 (Treestar, Ashland, OR). A list of antibodies used for flow cytometry is provided in Supplementary Table 3and gating strategies are shown in Supplementary Fig. 9.

Prior to tissue harvest, animals were anesthetized with isofluorane and intracardially perfused with cold heparinized saline. After removing the cerebellum and olfactory bulbs, brain tissue was processed immediately by fine cutting followed by digestion with an enzyme cocktail of collagenase/DNase (Sigma). Myelin was removed by density centrifugation in DMEM supplemented with 20% BSA, and the remaining cell pellet was resuspended in media for staining. Brains were processed individually, without pooling. Isolated cells were first blocked using CD16/CD32 Mouse BD Fc Block (BD Biosciences, San Jose, CA), and surface proteins were detected using monoclonal antibodies CD45 FITC (30-F11, Biolegend, San Diego, CA), CD11b BV510 (M1/70, Biolegend), CCR2 BV650 (SA203G11, Biolegend), CD3e PE (145-2C11, BD Pharmingen), CD19 PE-Cy7 (6D5, Biolegend), CD11c APC (N418, Biolegend), and Ly6c APC-Fire750 (HK1.4, Biolegend), CD74 BV421 (In1, BD). Dead cells were excluded using Live/Dead Fixable Blue (Thermo Fisher Scientific, Waltham, MA), according to manufacturer’s instructions. After staining, cells were washed and acquired using a FACSymphony (BD Scientific) equipped with FACS Diva v.8, and data were analyzed using FlowJo software v.10.0.7.