Magnetic-Activated Cell Sorting for the Fast and Efficient Separation of Human and Rodent Schwann Cells from Mixed Cell Populations
To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75NGFR, O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.
Key wordsPeripheral nerve Schwann cells Primary cultures Fibroblasts Magnetic bead separation p75NGFR Thy-1 O1 O4 Cell sorting
We are grateful for the technical assistance provided by Dr. Ketty Bacallao and Ms. Blanche Kuo. We thank Dr. James Guest for critically reviewing the manuscript. The work presented in this chapter was generously supported by the NIH-NINDS (NS084326), The Craig Neilsen Foundation (339576), The Miami Project to Cure Paralysis, and The Buoniconti Fund. The authors declare no conflicts of interest with the contents of this article.
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