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Toshio Hisatomi, Shoji Notomi, Atsunobu Takeda, Yasuhiro Ikeda, Tatsuro Ishibashi; High Biocompatibility Of BBG Depends On Low Cellular Permeability Into The Live Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2700.
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© ARVO (1962-2015); The Authors (2016-present)
We have reported Brilliant Blue G as a surgical adjuvant with good biocompatibility and staining ability of internal limiting membrane (ILM). To further examine the biocompatibility of BBG, we evaluate intracellular intake and accumulation of the dye. Since recently BBG was reported to be an antagonist of cellular surface P2X7 receptor, we tested possible contribution of P2X7 receptor to cellular staining of the dye.
We developed primary retinal cell culture from C57BL6 mouse. The culture was tested under normal and starvation condition without B27 and growth factors. Intracellular accumulation of Calsein AM and CMTMRos was analyzed for live cell imaging while propidium iodide for dead cell imaging. TUNEL was also stained for apoptotic cell death. To test the role of P2X7 receptor, P2X7 knockout mouse was also compared to wild type mouse under the same experiment.
Apoptotic cell death was increased under starvation condition. The live cells with positive staining for Calsein AM and CMTMRos barely stained with BBG. A part of TUNEL positive apoptotic cells and PI positive necrotic cells was positive for BBG. P2X7 receptor knockout mouse showed no remarkable changes on BBG staining compared to wild type mouse under starvation.
The live cells showed no staining of BBG, confirming substantial biocompatibility of the dye. The staining of apoptotic and necrotic cells was dependent on the cellular permeabilization after cell death rather than P2X7 receptor.
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