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P. Argueso, S. Spurr–Michaud, A. Tisdale, I.K. Gipson; Exclusion of Rose Bengal Staining in Stratified Islands of Cultured Human Corneal–Limbal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):74.
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Purpose: Rose bengal is an organic anionic dye commonly used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. Our aim was to evaluate rose bengal staining in the recently characterized human corneal–limbal epithelial (HCLE) cell line, known to produce and glycosylate membrane–associated mucins. Methods: HCLE cells were grown in a medium optimized for proliferation of keratinocytes (K–sfm, Gibco). After reaching confluence, cells were switched to DMEM/F12 supplemented with 10% calf serum and 10 ng/ml EGF, to promote stratification of corneal cells and their differentiation. Human corneal fibroblasts were grown in DMEM/F12 supplemented with 10% fetal bovine serum. To assess dye uptake, cell cultures were incubated for 5 min with 0.1% rose bengal in PBS and photographed. Western blot and immunofluorescence analysis for the membrane–associated mucin MUC16 and the mucin–associated H185 carbohydrate epitope on MUC16 in HCLE cells were performed using OC125 and H185 monoclonal antibodies, respectively. Results: In proliferating HCLE cells (lacking the expression of MUC16 and H185 carbohydrate), as well as human corneal fibroblasts (which do not express mucins), addition of rose bengal resulted in the staining of the cell cytoplasm and nucleus in all cells. As determined by Western blot, HCLE cells grown to confluence and switched to serum–containing media for 3 days stratified and produced MUC16 mucin and the H185 carbohydrate epitope. By immunofluorescence, MUC16 and H185 localized to apical cells found on islands of stratified regions of HCLE cell cultures. Rose bengal did not penetrate into the islands of stratified apical HCLE cells. In contrast, rose bengal stained all cells in 3 day–confluent cultures of corneal fibroblasts. Conclusions: These results indicate that stratification of corneal epithelial cells and their differentiation, as measured by the capacity to produce O–glycosylated membrane–associated mucins, provide protection against rose bengal penetrance in vitro and suggest a role for membrane–associated mucins and their oligosaccharides in the protection of the ocular surface epithelia.
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