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ACCEPTED MANUSCRIPT 2.11 Enzyme-Linked Immunosorbent Assay Cell culture media were assayed for IL-6 (#R000B, R&D Systems, MN, USA) and CCL2 (#ab100777, Abcam, Cambridge, UK) using a sandwich enzyme-linked immunosorbent assay (Cornish et al.) as per the manufacturer’s instructions. 2.12 Western blotting Whole cell protein lysates were extracted using the Cellytic M buffer (Sigma-Aldrich) containing a Protease Inhibitor Cocktail (Sigma-Aldrich). Western blotting was performed according to previously described methods with minor modifications (Begum et al., 2013; Walker and Steinle, 2007). 20μg of denatured protein was loaded onto a 4-20% Mini-Protean TGX Precast Protein gel (Bio-Rad, CA, USA) followed by semi-dry transfer to a nitrocellulose membrane. To measure the protein expression of COX5a in cells, a COX5a primary antibody (1:500-1:1000, #ab110262, Abcam) was used, as well as a secondary antibody-peroxidase conjugate for visualisation (Bio-Rad). The protein was visualised with chemiluminescence using a Clarity Western ECL kit (Bio-Rad) and images captured and analysed using a Chemidoc MP with Image Lab software (Bio-Rad). The expression of COX5a was normalized to GAPDH. 2.13 Mitochondrial membrane potential (∆Ψm) The JC-1 dye (Sigma-Aldrich) was used to assess changes in the mitochondrial membrane potential (∆Ψm) of living cells, following previously published methodology (Kokkinopoulos et al., 2013b; Smiley et al., 1991). Fluorescence of JC-1 (525nm/575nm) in cells was measured using flow cytometry (FACSort, LSRII) and analysed with FlowJo (FlowJo, OR, USA). Data is presented as a ratio of red to green fluorescence intensity. 2.14 Statistical analysis 11 ACCEPTED MANUSCRIPTPDF Image | Photobiomodulation with 670 nm light ameliorates retinal
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