in a minimal volume aCSF. After manually counting cell concentration using a Burker chamber, suspensions were further diluted to desired concentrations. For improved cell viability, the composition of aCSF was altered (NMDG-HEPES) for experiments using P60 mice (see Table S1) (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4, 0.5 CaCl2; adjusted to pH 7.4. To reduce debris when dissociating strongly myelinated regions, after filtering, the suspension was diluted in a large volume (15ml total) aCSF, followed by centrifugation, as above. Importantly, aCSF equilibrated in 95% O2 5% CO2 was used in all steps, and cells were kept on ice or at 4°C at all times except for enzymatic digestion.
