| Popis: |
An immunopanning technique for capturing the immediate progenitors of oligodendrocytes (OLs) that first appear In developing rat cerebrum at P4-P5 is described. Identified with two monoclonal antibodies, O4 and anti-galactocerebroside (GalC), these progenitors have the surface antigenic phenotype O4+GalC−. Their isolation from dissociated cell suspensions of tissue begins with a negative immunoselection step using antibody-mediated complement lysis to remove O4+ cells (15%) that have already differentiated to become GalC+ OLs at the time of dissociation. The remaining progenitors are labeled with monoclonal antibody 04, and 04+ cells are bound to a secondary antibody-coated polystyrene dish. After nonadherent cells are washed away, O4+GalC− progenitors are recovered and placed into primary culture. Yields of 3-4 × 105 progenitors per cerebrum are obtained with >90% viability and 95% purity. Culturing the progenitors in a defined, insulin-containing medium conditioned by type 1 astrocytes promotes extensive differentiation and long-term cell survival. Marked differences in the time course of progenitor accumulation and efficacy of immunopanning indicate that purification of O4+GalC− progenitors from regions other than cerebrum is necessary for some strains of mice. |
