Magnetic Affinity Cell Sorting (MACS) Separation and Flow Cytometric Characterization of Neural Cell Adhesion Molecule-Positive, Cultured Myogenic Cells from Normal and Dystrophic Dogs

Abstract

We developed a magnetic affinity cell sorting (MACS) assay based on differential expression of neural cell adhesion molecule (NCAM) isoforms in muscle cell cultures from normal and dystrophic dogs. NCAM is expressed during normal muscle differentiation, but has not been extensively examined within the context of muscle disease. A myogenic MACS assay could potentially maximize chances of obtaining normal nonsenescent, low-passage myogenic cells capable of proliferating in vitro and in vivo following transplantation. Myoblast-specific anti-NCAM polyclonal antibody directed against the NCAM isoform associated with muscle cell proliferation more effectively separated mixed canine cultures than did monoclonal antibodies directed against differentiated NCAM isoforms in the MACS assay. Flow cytometry using 5.1H11 anti-NCAM monoclonal antibody was then performed on normal and dystrophic fractionated cells and the results from these two groups were compared to each other and to nonfractionated cell populations. Normal canine cell cultures that had not been separated contained a larger percentage of FACscan-positive cells than did corresponding dystrophic canine cell cultures. Prior polyclonal anti-NCAM MACS separation of dystrophic cultures yielded higher numbers of adherent cells and higher gating percentages of 5.1H11-positive cell populations than did normal cultures. However, cells from dystrophic animals exhibited lower mean fluorescent expression of NCAM than normal cells.