Lysis binding buffer

Briefly, all cells but tonsils were incubated for 30 minutes at room temperature (RT) (tonsils on ice) in darkness. Sorting of B-cell subsets was performed using a FACSAria2 cell sorter (BD Biosciences, San Jose, CA) at RT. Compensation was automatically calculated by FACSDiva software using single-stained control samples with the mAbs previously listed. Immediately before acquisition, the cells were filtered through a 35-μm filter (Cell Stainer, BD Biosciences). The purity of the isolated B-cell subsets (>90%) was confirmed by sorting approximately 1,000 cells into PBS and reacquisition of the sorted B-cell subsets. The cells were sorted into 450 μl of lysis/binding buffer (Miltenyi Biotech, Bergisch-Gladbach, Germany), except for the first PBMNC processed, which were sorted into 1 ml of PrepProtect (Miltenyi Biotech, Bergisch-Gladbach, Germany). The sorted B-cell subsets were stored at -20°C.

Brains from 40 adult naïve C57BL/6 mice were extracted, pooled, dissociated using Accutase, and purified using 40% Percoll as described above. Cells were counted, and an aliquot was removed for control single stains and FMO tubes. Fc receptors were blocked, and live cells were labeled with Calcein Violet AM as before. Cells were then stained with a cocktail of anti-A2B5, PDGFRα, NG2, O4, GALC, and MOG in flow cytometry buffer for 30 min at 4°C. Cells were washed twice and resuspended in flow cytometry buffer. Early OPCs (A2B5⁺PDGFRα⁺), intermediate OPCs (O4⁺NG2⁺), and mature oligodendrocytes (GALC⁺MOG⁺) were sorted by FACS into lysis/binding buffer (Miltenyi Biotec) using a FACSAria 5 (BD Biosciences). Sorted cells were stored at −80°C. After thawing on ice, total RNA was isolated using µMACS mRNA Isolation Kit (Miltenyi Biotec) and cDNA was synthesized on column with µMACS One-step cDNA Synthesis Kit (Miltenyi Biotec). Gene expression was analyzed by qRT-PCR using specific primers (Qiagen, Valencia, CA, Table S2), RT² SYBR Green FAST Mastermix (Qiagen), and an iQ5 real-time PCR detection system (Biorad, Hercules, CA). Results were standardized to a housekeeping gene and normalized to gene expression in the A2B5⁺PDGFRα⁺ sorted early OPC population. Data is plotted as relative expression ± SD of technical replicates.