Methodologies to unlock the molecular expression and cellular structure of ocular lens epithelial cells.
PCR
RNA
Western blot
flat mount
immunostaining
primary culture
whole mount
Journal
Frontiers in cell and developmental biology
ISSN: 2296-634X
Titre abrégé: Front Cell Dev Biol
Pays: Switzerland
ID NLM: 101630250
Informations de publication
Date de publication:
2022
2022
Historique:
received:
30
06
2022
accepted:
16
08
2022
entrez:
30
9
2022
pubmed:
1
10
2022
medline:
1
10
2022
Statut:
epublish
Résumé
The transparent ocular lens in the anterior chamber of the eye is responsible for fine focusing of light onto the retina. The lens is entirely cellular with bulk of the tissue composed of fiber cells, and the anterior hemisphere of the lens is covered by a monolayer of epithelial cells. Lens epithelial cells are important for maintaining fiber cell homeostasis and for continual growth of the lens tissue throughout life. Cataracts, defined as any opacity in the lens, remain the leading cause of blindness in the world. Following cataract surgery, lens epithelial cells can undergo a process of epithelial-to-mesenchymal transition (EMT), leading to secondary cataracts due to posterior capsular opacification (PCO). Since the epithelial cells make up only a small fraction of the lens, specialized techniques are required to study lens epithelial cell biology and pathology. Studies using native lens epithelial cells often require pooling of samples to obtain enough cells to make sufficient samples for traditional molecular biology techniques. Here, we provide detailed protocols that enable the study of native mouse lens epithelial cells, including immunostaining of the native lens epithelium in flat mounts, extraction of RNA and proteins from pairs of lens epithelial monolayers, and isolation of lens epithelial cells for primary culture. These protocols will enable researchers to gain better insight on representative molecular expression and cellular structure of lens epithelial cells. We also provide comparative data between native, primary culture, and immortalized lens epithelial cells and discuss the advantages and disadvantages of each technique presented.
Identifiants
pubmed: 36176273
doi: 10.3389/fcell.2022.983178
pii: 983178
pmc: PMC9514789
doi:
Types de publication
Journal Article
Langues
eng
Pagination
983178Subventions
Organisme : NIGMS NIH HHS
ID : P20 GM139760
Pays : United States
Organisme : NEI NIH HHS
ID : R01 EY017724
Pays : United States
Organisme : NEI NIH HHS
ID : R01 EY032056
Pays : United States
Informations de copyright
Copyright © 2022 Parreno, Emin, Vu, Clark, Aryal, Patel and Cheng.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DS declared a shared affiliation with the author SA to the handling editor at the time of review.
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