Ravelo Kristine M, Andersen Natalia D, Monje Paula V
The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, USA.
Methods Mol Biol. 2018;1739:87-109. doi: 10.1007/978-1-4939-7649-2_6.
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 (p75, 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.
迄今为止，磁激活细胞分选（MACS）仍然是一种基于细胞表面差异标记来分离不同细胞群体的强大方法。本文介绍了用于细胞免疫标记的优化直接和间接MACS方案，作为去除雪旺细胞（SC）培养物中污染细胞（特别是成纤维细胞）并分离不同分化阶段SC群体的方法。本章描述了（1）从已建立的人和大鼠SC培养物中制备单细胞悬液，（2）使用SC特异性（p75、O4和O1）和成纤维细胞特异性（Thy-1）标记设计和应用细胞选择策略，以及（3）分选前后细胞群体的表征。提供了一种用于杂交瘤细胞培养生长的简单方案，作为用于SC和成纤维细胞表面免疫标记的单克隆抗体来源，作为市售产品的经济有效替代方案。这些步骤能够及时、高效地回收纯化的SC群体，同时不损害细胞的活力和生物学活性。
