Flavin adenine dinucleotide assay kit

Succinate dehydrogenase and catalase enzyme activity were measured in isolated mitochondria as previously described in detail [56 (link)]. Cytochrome c Oxidase Assay Kit and Citrate Synthase Assay Kit (Sigma-Aldrich/MERCK, Darmstadt, Germany) were used for determination of enzyme activities from 10 µg or 300 µg total protein of 11,000× g liver fractions, respectively, according to the manufacturer’s protocols. Membrane integrities to assess the quality of mitochondria preparations were measured with the Cytochrome c Oxidase Assay Kit (Sigma-Aldrich/MERCK, Darmstadt, Germany), according to the manufacturer’s protocol. NAD+, NADH, and FAD+ abundance were also measured in 11,000× g liver fractions, with 10 µg total protein from 11,000× g fractions used with the NAD+/NADH-Glo Assay (Promega, Germany) and 0.4 µg total protein from 11,000× g fractions used with the Flavin Adenine Dinucleotide (FAD+) Assay Kit (Abcam, Germany), according to the provided protocols from the manufacturers. A β-Hydroxybutyrat Assay Kit (Sigma-Aldrich/MERCK, Darmstadt, Germany) was used to measure the abundance of β-hydroxybutyrat in liver tissue, according to the manufacturer’s protocol.