Macs neural tissue dissociation kit p

Mouse OPCs were isolated from P6–P9 pups, as described previously (Watkins et al., 2008 (link); Swire and ffrench-Constant, 2019 (link)). Ear clips were taken for subsequent genotyping. Briefly, cerebral cortices were dissected, diced, and dissociated into single-cell suspensions gently using MACS Neural Tissue Dissociation Kit P (catalog #130–092-628, Miltenyi Biotec). Cells were resuspended in 0.2% BSA, insulin, and PBS, and were transferred to treated tissue culture dishes coated with BSL1 (catalog #L-1100, Vector Laboratories) twice for 15 min. Cell solutions were then transferred to dishes coated with anti-PDGFRα (CD140a) for 45 min. Solutions were aspirated, and attached cells were washed twice with media and removed with a cell scraper. All collected cells were added to vented T75 flasks and grown at 37°C with 7.5% CO₂. Cells were grown in myelination media containing PDGF and neurotrophin-3, were changed every 2 d, and were supplemented daily with PDGF. After 7–9 d, confluent flasks were washed with PBS and then detached using TrypLE for 10 min at 37°C. Solutions were centrifuged at 1000 rpm for 5 min, resuspended, and counted using a hemocytometer.

Cell cultures were dissociated using Accutase® (Sigma-Aldrich). Xenografts were dissociated with MACS Neural Tissue Dissociation Kit (P) (Miltenyi) following the manufacturers’ instructions. Single cells were resuspended in HBSS, 2% FBS, 10 mM HEPES buffer (100 µl per test). Cells were incubated with the IR-LIVE/DEAD® Fixable Dead Cell Stains (Invitrogen; 1 µg ml⁻¹) and appropriate preconjugated antibodies for 30 min at 4 °C in the dark (Supplementary Table 3). For cell cycle analysis in viable cells, cells were prestained with Hoechst 33342 (5 µg ml⁻¹, Bisbenzimide, Ho342; Sigma) at 37 °C before antibody staining^{37 (link)}. Data acquisition was performed on a FACS Aria^TM SORP cytometer (BD Biosciences) and ImageStream imaging cytometer (Amnis). Data acquisition and analysis were done for FACSAria with DIVA software (BD Bioscience); and INSPIRE and IDEAS^® for ImageStream. Histograms were prepared with the FlowJo software.

Flow cytometry experiments were performed as described before [16 (link), 17 (link)]. Briefly, xenografts derived in eGFP-expressing mice were minced with scalpels and dissociated with MACS Neural Tissue Dissociation Kit (P) (Miltenyi, 130-092-628, Lund, Sweden) following the manufacturer’s instructions. Single cell suspensions were incubated with Hoechst 33342 (5 µg/ml, Bisbenzimide, Ho342; Sigma) at 37 °C in pre-warmed DMEM, containing 2 % FBS, 10 mM HEPES pH 7.4 and DNAse I (10 µg/ml; Sigma) at 1 × 10⁶ cells/ml for 120 min. After washing, cells were resuspended in ice-cold HBSS 2 % FBS and 10 mM HEPES pH 7.4 buffer (100 µl/test). Prior to flow cytometry, cells were incubated with LIVE/DEAD^® Fixable Dead Cell Stains (Life Technologies) and appropriate preconjugated antibodies for 30 min at 4 °C in the dark (antibodies are listed in Supplementary Table II). Data acquisition was performed on a FACS Aria™ SORP cytometer (BD Biosciences, San Jose, CA, USA) and the Hoechst signal was excited with the UV laser. Data acquisition and analysis were done with DIVA software (BD Biosciences). Histograms were prepared with the FlowJo software.