For our experiments, HCEC cells will be seeded onto microplates from the manufacturer and grown to confluence. Then the cells will be treated with butyl hydroperoxide with and without n-acetylcysteine through standard injection ports. Assays will be initiated by replacing the growth medium with XF assay medium, which is formulated with unbuffered Dulbecco's modified Eagle's medium, 2 mM sodium pyruvate and 25 mM glucose, pH 7.4. The mitochondrial assays will include oligomycin (to inhibit ATP synthase activity), tri-fluorocarbonylcyanide phenylhydrazone (FCCP) (to uncouple mitochondrial oxidative phosphorylation), and rotenone and antimycin A (to inhibit electron transport in complex I and III, respectively). Subsequently, data can be analyzed to obtain mitochondrial reserve capacity among a host of many other metabolic parameters (**). After we establish baseline data with normal HCEC, we will repeat experiments with HCEC derived from FECD corneas. Our next step will be to assess if the mitochondrial inhibitors employed in the experiments above affect PAMR and TER (as measured by
For our experiments, HCEC cells will be seeded onto microplates from the manufacturer and grown to confluence. Then the cells will be treated with butyl hydroperoxide with and without n-acetylcysteine through standard injection ports. Assays will be initiated by replacing the growth medium with XF assay medium, which is formulated with unbuffered Dulbecco's modified Eagle's medium, 2 mM sodium pyruvate and 25 mM glucose, pH 7.4. The mitochondrial assays will include oligomycin (to inhibit ATP synthase activity), tri-fluorocarbonylcyanide phenylhydrazone (FCCP) (to uncouple mitochondrial oxidative phosphorylation), and rotenone and antimycin A (to inhibit electron transport in complex I and III, respectively). Subsequently, data can be analyzed to obtain mitochondrial reserve capacity among a host of many other metabolic parameters (**). After we establish baseline data with normal HCEC, we will repeat experiments with HCEC derived from FECD corneas. Our next step will be to assess if the mitochondrial inhibitors employed in the experiments above affect PAMR and TER (as measured by