Primary mixed glia cultures
Brains from 8-week-old C57BL/6 J mice (Charles River) were collected and transported in Hank’s Balanced Salt Solution (HBSS without Mg2+ or Ca2+; Gibco) on ice. These culture experiments were repeated 10 times (n = 10), 4 were used to generate RNA-Sequencing replicates and 6 for subsequent validation experiments. For most cultures three brains were sufficient per experiment (one 6 well-plate), though for some experiments more cells were required so cultures were scaled up accordingly. Brains were digested using the Neural Tissue Digest Kit-P in C-tubes as per manufactures directions (Miltenyi Biotec). Two brains were digested in each C-tube using program 37_ABDK on the GentleMACS OctoDissociator (Miltenyi Biotec) to produce a single cell suspension. Cell suspensions were passed through a 70 μM strainer (Falcon) into a 50 mL tube and centrifuged at…
