Spectramax m5 multi functional plate reader

Advanced Glycation End Product concentrations were determined using the OxiSelect™ Advanced Glycation End Product (AGE) ELISA kit (Cell Biolabs, Inc. San Diego, CA) following manufacturer’s instruction. AGEs were detected using an anti-AGE antibody with a HRP-conjugated secondary antibody and detected spectrophotometrically at 450 nm. Absorbances were measured by the SpectraMax M5 Multi-Functional Plate Reader (Molecular Devices, Sunnyvale, CA). Data are presented as μg/mL.

Reactive oxygen species production was measured by the OxiSelect™ Protein Carbonyl ELISA kit (Cell Biolabs, Inc.), an indirect measurement of ROS production. This assay involves derivitization of the carbonyl group with dinitrophenylhydrazine (DNPH), followed by detection with a anti-DNP antibody detection, which can be measured spectrophotometrically at 375 nm (Reznick and Packer 1994 (link); Talent et al. 1998 (link)). Oxidized BSA served as the standard control. Confluent samples were incubated in the respective oxygen tensions for 4 days. Cells were then sonicated in lysis buffer (25 mmol/L HEPES, 150 mmol/L NaCl, 10 mmol/L MgCl₂, 1 mmol/L EDTA, 2% glycerol, pH 7.5), and protein concentration was determined by BCA protein assay (Pierce Protein Research Products, Rockford, IL). Absorbances were measured by the SpectraMax M5 Multi-Functional Plate Reader (Molecular Devices).