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After 24 h, cells were lysed and subjected to luciferase activity using a Luc-Pair Duo-Luciferase HS Assay Kit (GeneCopoeia, MD, USA) according to the manufacturers instructions
Cell pellets (~2030 l of packed cell pellet) were re-suspended in 1 ml buffer containing 20 mM HEPES (pH = 7.5), 10 mM KCl, 250 mM sucrose, 5 mM MgCl 2 , 1 mM EGTA, 1x phosphatase inhibitor cocktail (Sigma, P0044) and 1x protease inhibitor cocktail (Sigma, P8340), and lysed by addition of digitonin (Sigma, D141), 200 g/ml final concentration at 4 C for 10 min
However, previous studies have not resolved certain questions: i) whether alterations in the identified proteins and metabolites are affected by eye drops, ii) how these changes in proteins and metabolites are affected by glaucoma phenotype, and iii) whether normal-tension glaucoma shows protein and metabolite changes in the same way as POAG