Isolation and Differentiation of Oligodendrocyte Progenitor Cells

Mixed glial cultures generated from P0 to P3 neonatal wild-type mice were maintained in full DMEM for 10 to 14 days until a monolayer of astrocytes on the bottom and primary oligodendroglial progenitor cells (OPCs) with loosely attached microglia on the top, were apparent. The separation of OPCs was achieved initially with the removal of microglia, by shaking in 200 rpm for 1 h in 37 °C and then with continuous shaking under the same conditions for 18 h, as previously described^{56 (link)}. Afterwards, isolated cells were platted on poly-D-lysine-coated coverslips (P7405, Sigma-Aldrich, USA) with a density of 80,000 cells/mm² and maintained in SATO medium (284369) supplemented with Insulin-Transferrin-Selenium solution (41400045, ITS- Gibco, Invitrogen, Carlsbad, CA, USA), 1% penicillin/streptomycin and 1% horse serum (H1138; Sigma-Aldrich, St. Louis, MO, USA) for 4 days. αSyn fibrils (final concentration 0.5 µg/mL culture medium/well) amplified from human MSA and PD brains were added to TPPP/p25α-positive mature differentiated oligodendrocytes for 48 h and then cells were fixed and preceded for immunofluorescence analysis. All experimental procedures were approved by the Ethics Committee for the Use of Laboratory Animals in the Biomedical Research Foundation of Athens.