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J.R. Sabah, B.D. Schultz, Z.W. Brown, J. Reddan, L.J. Takemoto; Transcytotic Passage of Albumin Through Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4102.
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To characterize the possible transcytotic passage of albumin through lens epithelial cells.
N/N 1003A rabbit lens epithelial cells were grown to confluence on Transwell semi–permeable membranes supports. After 1 week in culture, cells were switched to serum–free DMEM medium for 2 hr, and then exposed to DMEM supplemented with albumin (5 mg/ml) for 30 min prior to experimentation. Experiments were conducted in the absence and presence of endocytic inhibitors (filipin, 1.25 µg/ml or dansylcadaverine, DCV, 6.5 µg/ml). Alexa 488–albumin (2.5 mg/ml) was added to the upper (apical) Transwell chamber at the outset of the experiment and transcytotic passage of albumin was monitored every hour for 4 hr by quantifying fluorescence in the lower (basolateral) Transwell chamber.
Treatment with filipin or DCV reduced the passage of Alexa 488–albumin through lens epithelial cells by 32% and 37%, respectively. Z–stack reconstruction by confocal microscopy showed that the protein passage through the cell monolayer was predominantly transcellular.
The Transwell apparatus can be used to characterize passage of macromolecules such as albumin across monolayers of N/N lens epithelial cells. Albumin passage, which relies on an endocytic step, can be partially inhibited by filipin and dansylcadaverine, suggesting the possible involvement of caveolae and clathrin–coated vesicles in the transcytotic process.
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