Cultures of oligodendrocyte precursor cells (OPCs) were prepared by immunopanning and grown as outlined in Methods. To measure proliferation, OPCs were grown for 24 hours in OPC proliferation media47 and then changed into OPC media containing 10μM EdU (ThermoFisher, C10339) and varying concentrations of A1 or resting ACM (0–50 μg/ml total protein). After 5 days, the cells were fixed, permeabilized, and stained for EdU and DNA (Hoechst 33342) according to the protocol for the Click-It® Edu Imaging Kit. To measure differentiations of OPCs into mature OLs, 1ug/ml A1 ACM was added to OPC cultures and they were imaged at 24 h intervals with phase time lapse microscope (IncuCyte Zoom ® System). Images were analyzed and number of primary processes extending from the cell soma were counted. A cell was considered an OPC with 0–2 processes, a differentiating OL with 4–5 processes, and a mature OL with 5+ primary processes. Before differentiation into mature OLs, OPC migration was measured using the Template Matching and Slice Alignment and MTrackJ plugins for ImageJ. Astrocyte motility was measured using the same ImageJ plugin, with cells grown at a density of 5000 cells/cm2 in HBEGF-containing astrocyte growth media.
