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

Yuzuru Sasamoto; Philip C. Yeung; Johnathan Tran; Markus H. Frank; Natasha Y. Frank

STAR Protocols (Sep 2023)

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

Yuzuru Sasamoto,
Philip C. Yeung,
Johnathan Tran,
Markus H. Frank,
Natasha Y. Frank

Affiliations

Yuzuru Sasamoto: Division of Genetics, Brigham and Women’s Hospital, Boston, MA, USA; Transplant Research Program, Boston Children’s Hospital, Boston, MA, USA
Philip C. Yeung: 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
Johnathan Tran: Transplant Research Program, Boston Children’s Hospital, Boston, MA, USA
Markus H. Frank: Transplant Research Program, Boston Children’s Hospital, Boston, MA, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA; Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women’s Hospital, Boston, MA, USA; School of Medical and Health Sciences, Edith Cowan University, Perth, WA, Australia; Corresponding author
Natasha Y. Frank: Division of Genetics, Brigham and Women’s Hospital, Boston, MA, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA; Department of Medicine, VA Boston Healthcare System, Boston, MA, USA; Corresponding author

Journal volume & issue: Vol. 4, no. 3
p. 102503

Abstract

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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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