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通过流式细胞术和细胞分选分离人 BCAM 阳性角膜祖细胞的方案。

Protocol for isolating human BCAM-positive corneal progenitor cells by flow cytometry and cell sorting.

机构信息

Division of Genetics, Brigham and Women's Hospital, Boston, MA, USA; Transplant Research Program, Boston Children's Hospital, Boston, MA, USA.

Division of Medical Sciences, Harvard Medical School, Boston, MA, USA; Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.

出版信息

STAR Protoc. 2023 Sep 15;4(3):102503. doi: 10.1016/j.xpro.2023.102503. Epub 2023 Sep 3.

DOI:10.1016/j.xpro.2023.102503

PMID:37669162

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10485628/

Abstract

BCAM-positive basal limbal epithelial cells are an early transit-amplifying cell population (TAC) capable of holoclone formation and corneal epithelial differentiation. Here, we present a protocol for isolating BCAM-positive cells from human donor corneas by flow cytometry and cell sorting. We describe steps for cell dissection and dissociation, antibody staining, and flow cytometry. We then detail procedures for culturing the purified BCAM-positive and BCAM-negative cells for holoclone and cell sheet formation assays to study the factors that regulate corneal regeneration. For complete details on the use and execution of this protocol, please refer to Sasamoto et al..

摘要

BCAM 阳性基底边缘上皮细胞是一种早期过渡扩增细胞群（TAC），能够形成全克隆和角膜上皮分化。在此，我们通过流式细胞术和细胞分选从人供体角膜中分离 BCAM 阳性细胞的方案。我们描述了细胞解剖和分离、抗体染色和流式细胞术的步骤。然后，我们详细介绍了培养纯化的 BCAM 阳性和 BCAM 阴性细胞进行全克隆和细胞片形成测定的程序，以研究调节角膜再生的因素。有关此方案的使用和执行的完整详细信息，请参阅 Sasamoto 等人的研究。