Preparation and culture of rat lens epithelial explants for studying terminal differentiation
| Autor: | Senthil S. Saravanamuthu, Chun Y. Gao, Peggy S. Zelenka |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Pathology
medicine.medical_specialty lens General Chemical Engineering Biology Fibroblast growth factor General Biochemistry Genetics and Molecular Biology law.invention Tissue Culture Techniques 03 medical and health sciences Tissue culture 0302 clinical medicine Ciliary body law Lens Crystalline medicine Animals FGF rat Cells Cultured Issue 31 030304 developmental biology 0303 health sciences Retina General Immunology and Microbiology General Neuroscience Cell Differentiation Epithelial Cells differentiation Epithelium Lens Fiber Cell biology Rats Lens (optics) Cellular Biology medicine.anatomical_structure 030221 ophthalmology & optometry Fibroblast Growth Factor 2 Explant culture |
| Zdroj: | Journal of Visualized Experiments : JoVE |
| ISSN: | 1940-087X |
| Popis: | The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body. Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens. Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2. Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers. The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100 ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied. Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques. Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways. |
| Databáze: | OpenAIRE |
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