An improved method for high-purity isolation of oligodendrocytes from neonatal and adult mouse brain.

Oligodendrocytes are central to myelination and are key targets in myelin disease research and gene therapy. We optimized magnetic isolation of O4+ oligodendrocytes from adult mouse brain and PDGFRα+ oligodendrocyte precursor cells from neonatal brain, and evaluated purity, viability, maturation, metabolism, and rAAV transduction. The protocol yielded highly enriched adult O4+ cells and neonatal OPCs with preserved viability, structural integrity, and developmental potential. Isolated adult cells maintained oxidative and glycolytic activity, incorporated glucose carbon into central metabolic pathways, and supported the quantification of oligodendrocyte-directed rAAV9-phMBP-EGFP transduction. Neonatal PDGFRα+ cells proliferated and differentiated into MBP- and QKI7-positive oligodendrocytes. This approach provides a practical platform for studying oligodendrocyte biology and evaluating therapeutic vectors and other interventions in myelin disorders.