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Mariusz Zareba, Patrycja Kaczara, Christine Skumatz, Tadeusz J. Sarna, Magdalena Olchawa, Anna Pilat, Janice M. Burke; RPE Melanosomes Protect Cells against Non-Photic Stress. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4424.
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© ARVO (1962-2015); The Authors (2016-present)
Melanosomes are believed to protect RPE cells against oxidative stress due to light exposure, and may theoretically also protect against other sources of stress because of the antioxidant properties of melanin. Here we developed two dynamic cell death assays to determine whether melanosomes affect survival in cultured cells treated with H2O2 to induce non-photic stress.
ARPE-19 cells contained either phagocytized porcine melanosomes or control non-specific particles (latex beads). Two methods for H2O2 delivery were used (pulse delivery or continuous generation with glucose oxidase [GOx]) followed by two dynamic assays to evaluate cell death over time using nuclear propidium iodide fluorescence as a cell death reporter. In one assay, cell death was quantified in the entire cell population; in the other, death in cell sub-populations pre-selected for their particle content was quantified by live cell imaging.
ARPE-19 cells containing melanosomes were more resistant to H2O2-induced stress than cells containing beads. The differential sensitivity was detected by comparing paired cultures of cells containing different particle types and, using the imaging method, by comparing adjacent cells in the same co-cultures pre-loaded with different particle types. Particle-mediated stress resistance was observed both following pulse delivery and with enzymatic generation of H2O2. It manifest as a delayed time of death onset and a slower recruitment of cells into the susceptible population over time. Photobleaching of melanosomes by light irradiation before phagocytosis, a protocol that simulates age-related melanin photo-oxidation, diminished the stress resistance conferred by the pigment granules.
The dynamic assays used here may have utility for detecting small pro- or antioxidant effects in cultured cells, and the results that were obtained indicate that pigment granules perform functions within RPE cells aside from those related to light irradiation.
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