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ACCEPTED MANUSCRIPT in the same incubator, but shielded with aluminium foil to avoid light exposure. For air/gas exchange, small incisions were cut on the aluminium foil. Following 4.5 hours of PD, co- cultures were incubated under dim light conditions with 5% CO2 at 37°C. After 24 hours of recovery, cells and supernatant were collected for analysis. 2.5 670nm red light treatment of co-cultures The 670nm LED array (Quantum Devices) was applied to different co-culture groups (with or without PD stress) as follows. (A) PD + 670nm group - inserts containing 661W cells were removed from the co-culture during 670 nm light exposure. Only Müller cells were 2 exposed to 670nm light (9J/cm ) and were treated three times over the first 12 hours of recovery following PD. (B) Control + 670nm group - Müller cells received treatment with 670nm light using the same paradigm, but with no PD. For (C) PD only and (D) control groups, the inserts were removed from plates and only Müller cells were exposed to the 670nm LED array, but with the light source switched off. 2.6 Isolation and assessment of activation of rat MG/MΦ Rat retinal microglia and macrophages (MG/MΦ) from SD rats (P50-60) were isolated using a fluorescence-activated cell sorter (FACS) (BD FACSAria II; BD Biosciences, NJ, USA), using previously described protocols with minor modifications (Fernando et al., 2016; Ma et al., 2013; Rutar et al., 2015). Isolated cells were subsequently cultured in GM containing mouse granulocyte-macrophage colony-stimulating factor (GM- CSF, 1ng/ml; Stem Cell Technologies, Vancouver, Canada) with 5% CO2 at 37°C. Media was replaced every 3-4 days until cells reached 80% confluency. Supernatant (SN) was collected from co-cultures of the control, 670nm only, PD only and PD+670nm groups. MG/MΦ cells were incubated with the SN of these groups for 24 8 ACCEPTED MANUSCRIPTPDF Image | Photobiomodulation with 670 nm light ameliorates retinal
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