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

• DOI: 10.1016/j.xpro.2023.102503
• Publication Date: 2023-09-03T21:28:40Z
• Authors: Yuzuru Sasamoto; Philip C. Yeung; Johnathan Tran; Markus H. Frank; Natasha Y. Frank
• Keywords: Q1-390; Science (General); Stem Cells; Medicine and Health Sciences; Protocol; Cell Isolation; Cell Biology; Cell-based Assays; Flow Cytometry/Mass Cytometry

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.1.