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PT Carman, L Takemoto, DL Boyle; Methodology for the Delivery of Various Macromolecules into Cells of the Intact Lens . Invest. Ophthalmol. Vis. Sci. 2002;43(13):464.
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Purpose: To develop a non-invasive method for the delivery of macromolecules into cells of the intact lens. Methods: Adult rat eyes were enucleated and intact lenses were dissected from the eye and placed in 0.25 ml Minimum Essential Medium Eagles with non-essential amino acids, Earle's salts, L-glutamine, phenol red and sodium bicarbonate. Texas Red sulfonyl chloride was conjugated to alpha, beta, or gamma crystallins according to the manufacture's protocol (Molecular Probes). Dextran (10kDa and 70 kDa) conjugated Texas Red was purchased from Molecular Probes. Macromolecules were incubated with lenses for various times at 37 degrees Celsius, with and without prior encapsulation into cationic lipid vesicles. Introduction of a macromolecule conjugated fluorophore into cells of the intact lens were determined by standard laser scanning confocal microscopy. Results: All the macromolecules tested were delivered into cells within the intact lens with and without prior incorporation into cationic lipid vesicles. Sytox Green (Molecular Probes) was excluded from labeling nuclei of cells in the intact lens. Conclusions: We have developed non-invasive methodologies for the delivery of macromolecules into cells of the intact lens. These methodologies should facilitate future studies on functions and behaviors of given macromolecules within cells of the intact lens.
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