Ghost red 780 viability dye

Lymph nodes and spleens were harvested in RPMI and pressed through a 70 μm filter to achieve a single cell suspension for compensation control samples. Brains were harvested and manually homogenized using a dounce homogenizer as previously described (12 (link)). Brain samples were filtered through a 70 μm filter to achieve a single cell suspension into a 50% percoll solution. Samples were centrifuged at 7,840 g. The myelin debris layer formed at the top of the gradient was aspirated. All samples were washed twice and plated in a 96-well v bottom plate. Peptide:MHC tetramers were constructed by our lab and samples were stained at a 1:100 dilution of tetramer for 30 min at room temperature in the dark. Following a wash, antibodies against CD45, CD11c, CD11b, I-A^b, CD80, CD86, VEGFR2, Nrp-1, CD4, CD8α, PD-1, and Tim-3 were used for staining at a 1:100 dilution for 30 min on ice in the dark (BD Biosciences, San Jose, CA; Tonbo Biosciences, San Diego, CA) in addition to Ghost Red 780 Viability Dye used at a 1:1000 dilution (Tonbo Biosciences, San Diego, CA). Samples were fixed with 2% paraformaldehyde. Samples were subsequently run on a BD LSRII flow cytometer equipped with FACSDiva software (BD Biosciences, San Jose, CA). Samples were digitally compensated using single-stained controls and analyzed by FlowJo v10 software (FlowJo LLC, Ashland, OR).