Fluorescence-activated cell sorter analysis of bulk-isolated porcine oligodendrocytes

We describe here the methods by which enriched populations of oligodendrocytes were isolated from adult porcine brains using Percoll density gradient centrifugation and their immunological properties analyzed by a fluorescence-activated cell sorter (FACS). The analyses by immunofluorescence microscopy and FACS indicated 95% and 93% purity of oligodendrocytes in the cell preparations when galactocerebroside antibody, an oligodendrocyte-specific marker, was employed. Further FACS analysis indicated that 90% of the cells with the forward angle light scatter characteristics of oligodendrocytes were immunolabelled by the monoclonal antibody HNK-1. The FACS sorting capability was then used to correlate the forward angle light scatter and propidium iodide uptake characteristics of our cell preparations to cell debris, erythrocytes as well as viable and non-viable oligodendrocytes. By gating on a multiparameter basis on both defined forward light scatter and positive galactocerebroside immunolabelling, oligodendrocyte populations exceeding 98% purity were obtained which were suitable for long-term culture and further immunological and neurobiological studies.