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ACCEPTED MANUSCRIPT pellets were collected by spinning at 210g for 10 minutes at 37°C and then dissociated in fresh GM by triturating with plastic serological pipettes. Cell suspension was cultured in a T25 flask (Thermo Fisher Scientific) in a humidified incubator with 5% CO2 at 37°C. The GM was left unchanged for the initial 4 days; GM was replenished on day 5. After 7-8 days, cellular aggregations, which are attached to the base of the flask, were removed by agitating the media and the GM was replenished. Until cells reached 90% confluency, cells were detached with 0.25% trypsin/EDTA from a T25 flask and then expended in a T75 flask (Thermo Fisher Scientific). After reaching 90% confluency in a T75 flask, cells were subcultured in the appropriate plates for subsequent experiments. Primary Müller cell characteristics were confirmed using immunolabelling for vimentin, S100β, glutamate synthetase (GS) (data not shown). 2.4 Photo-oxidative damage in co-culture of 661W cell line with primary Müller cells For mimicking in vivo interactions between photoreceptors and Müller cells, a transwell co-culture system was used. Primary Müller cells were seeded into 24-well plates at 45 a density of 2.5x10 cells per well or 6-well plates at density of 2x10 cells per well in GM, and plates incubated in 5% CO2 at 37°C. After 24 hours, in separate plates, 661W cells were seeded onto the membranes of transwell inserts (pore size 0.4μm; Corning, NY, USA) at a 34 density of 4x10 cells per insert (24-well transwell), or at a density of 5x10 cells per insert (6-well transwell) in GM, and were incubated with 5% CO2 at 37°C for 24 hours. Following incubation, inserts containing 661W cells were placed into wells seeded with Müller cells. The co-cultures were incubated in reduced-serum DMEM (supplemented with 1% FBS, L- 2 glutamine and antibiotic-antimycotic) and exposed to 15,000 lux light (2.2mW/cm ; irradiance measured with PM100D optical power meter, THORLABS, NJ, USA) from two white fluorescent lamps (2x10W T4 tri-phosphor 6500K daylight fluorescent tubes; Crompton, NSW, Australia), for 4.5 hours with 5% CO2 at 37°C. Control plates were placed 7 ACCEPTED MANUSCRIPTPDF Image | Photobiomodulation with 670 nm light ameliorates retinal
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