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ACCEPTED MANUSCRIPT hours with 5% CO2 at 37°C. MG/MΦ were then collected for RNA extraction, or immunostained with CD11b/CD86 for flow cytometry. MG/MΦ were collected and fixed in 2% paraformaldehyde (PFA) for 10 minutes on ice. Cells were blocked with 1% BSA and then incubated with biotin CD86 (1:100; Biolegend), or anti-rat CD11b PE antibody in 1% BSA for 30 minutes on ice. Cells were washed once in PBS containing 0.2% Tween-20 and resuspended in the secondary antibody with streptavidin conjugated with Alexa-Fluor-488 (S32453; Thermo Fisher Scientific) for 30 minutes on ice. The expression of CD11b or CD86 was measured using FACSort (LSRII; BD, CA, USA) and data was analysed using FlowJo (FLOWJO, OR, USA). 2.7 Assessment of cell viability and cell toxicity For measuring cell viability, an ATPlite 1 step assay (PerkinElmer, MA, USA) and an MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; Sigma-Aldrich) were used according to the manufacturer’s instructions and protocols previously described (Lu et al., 2013; Natoli et al., 2016). A CellTox assay (Promega, Madison, WI, USA) was performed to measure cell death according to previously described methods (Natoli et al., 2016). 2.8 Immunocytochemistry on primary Müller cells Müller cells were seeded onto poly-L-lysine-coated (100μg/ml; Sigma-Aldrich) glass 42 coverslips at a density of 30x10 cells/mm for 48 hours and then co-cultured with 661W cells with/without 670nm light as described above. Coverslips containing Müller cells were fixed with 2% PFA for 30 minutes and then washed twice with PBS. Immunocytochemistry was performed as described previously (Albarracin et al., 2011; Albarracin and Valter, 2012) using a primary antibody for COX5a (1:500, #ab110262, Abcam), an anti-mouse IgG (H+L) conjugated with biotin (SAB3701153; Sigma-Aldrich) and Streptavidin conjugated with 9 ACCEPTED MANUSCRIPTPDF Image | Photobiomodulation with 670 nm light ameliorates retinal
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