Protein carbonyl colorimetric kit

The protein carbonyl content in co-cultured cells was evaluated using the Protein carbonyl colorimetric kit (Cayman Chemical, Ann Arbor, MI, USA), based on the spectrophotometric detection of the protein hydrazone as product of the reaction between 2.4-dinitrophenyl hydrazine with protein carbonyls. The protein concentrations of samples were determined using the Pierce method (Pierce BCA Protein Assay Kit, ThermoFisher Scientific, Rockford, IL, USA). The absorbance of samples was determined at a test wavelength of 370 nm using an ELISA microplate reader (Varioskan™ LUX, ThermoFisher Scientific, Rockford, IL, USA). All experiments were performed in three independent replicates. The results were expressed as carbonyl content (nmol/mg) = (carbonyl nmol/mL)/(protein mg/mL).

The activities of reduced (glutathione, GSH) and oxidised (glutathione disulphide, GSSG) glutathione in the diaphragm were measured using glutathione fluorescent detection kit (DetectX K006-F1, Arbor Assays, MI, USA) and expressed as GSH:GSSG ratio.
The 20 S proteasome levels in the diaphragm were measured fluorometrically in total protein extracts using an assay kit (BML-AK740 assay kit, Enzo Life Sciences, NY, USA). The specific activity of the proteasome was calculated according to kit instructions and normalised against total protein concentration.
The protein carbonyl content in diaphragm was measured using a commercially available protein carbonyl colorimetric kit (Cayman, Ann Arbor, MI, USA).

Levels of total GSH from tissues were quantified using a fluorimetric method (#CS1020; Glutathione Assay Kit, Fluorimetric; Sigma, St. Louis, MO, USA). Oxidative stress was assessed by measuring the quantification of protein carbonyls and MDA using a Protein Carbonyl Colorimetric Kit and TBARS Assay kit (Cayman Chemical, Ann Arbor, MI, USA). Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (ThermoFisher Scientific, Grand Island, NY, USA) was used to detect hydrogen peroxide (H₂O₂) and expressed as nmol/mg protein. Protocols, as provided in the manufacturer’s instructions, were followed in all the assays using the appropriate controls and standards. Plasma and cell culture supernatant levels of irisin were determined using ELISA kits from Phoenix Pharmaceuticals, Inc. Burlingame, CA, USA (Cat# EK-067-29).