Complex 2 enzyme activity microplate assay kit

Protein lysates were analyzed for complex I abundance using the NADH dehydrogenase Human SimpleStep^TM ELISA kit (Abcam, ab178011), for complex IV abundance using the cytochrome c oxidase Human SimpleStep^TM ELISA kit (Abcam, ab179880) and for complex V using the ATP synthase Human Profiling ELISA kit (Abcam, ab124539). Mitochondrial respiratory complex activity was determined using the Complex I enzyme activity microplate assay kit (Abcam, ab109721), the Complex II enzyme activity microplate assay kit (Abcam, ab109908) and the Complex IV human enzyme activity microplate assay kit (Abcam, ab109909). Protein abundance or activity were determined using the linear range of a standard curve made with HeLa cells lysates (complexes I and IV abundance), with HepG2 cells lysate (complex V abundance) or bovine heart mitochondrial lysate (BHM) (Abcam, ab110338) (complexes I, II and IV activity). All assays were performed following the manufacturer’s instructions. In addition, all assays were validated to ensure that the signal was in the linear range of the detection system.

The mitochondrial membrane potential (MMP) was measured using MitoProbe™ JC-1 Assay Kit (#M34152, Thermo Fisher Scientific, Waltham, MA) as previously described [15 (link)]. The activities of complexes on the electron transport chain were measured using Complex I Enzyme Activity Microplate Assay Kit (Abcam, #ab109721); Complex II Enzyme Activity Microplate Assay Kit (Abcam, #ab109908); and Complex IV Human Enzyme Activity Microplate Assay Kit (Abcam, #ab109909) according to the manufacturers’ protocol. Results were normalized by the protein amounts in each assay.

The mitochondrial respiratory enzyme activities such as NADH dehydrogenase (Complex I Enzyme Activity Microplate Assay Kit, Abcam Trading Company Ltd. Shanghai, China.), succinate dehydrogenase (Complex II Enzyme Activity Microplate Assay Kit, Abcam Trading Company Ltd. Shanghai, China) Enzyme Activity Microplate Assay Kit, Abcam Trading Company Ltd. Shanghai, China) and cytochrome oxidase (cytochrome c oxidase assay kit, Sigma-Aldrich Co, St Louis, MO, USA) were estimated according to manufacturer’s instruction.

Mitochondrial respiratory chain complex I II, III, and IV were measured by Complex I Enzyme Activity Microplate Assay Kit (ab109721, abcam, Cambridge, United Kingdom), Complex II Enzyme Activity Microplate Assay Kit (ab109908, abcam, Cambridge, United Kingdom), MitoTox Complex II + III OxPhos Activity Assay Kit (ab109905, abcam, Cambridge, United Kingdom), and Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911, abcam, Cambridge, United Kingdom) respectively according to the manufacturer’s recommendation. Briefly 80 μg mitochondrial proteins were loaded in a 96-well plate, and the enzyme activity was determined colorimetrically with BioTek Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, United States) by detecting the light absorption value of the sample at 450 nm (complex I), 600 nm (complex II), and 550 nm (complex III and complex IV) respectively.

For a Caspase-3 Assay Kit (ab39401, Abcam), infarct tissue was immersed in 400 μl of lysis buffer and disrupted using a homogenizer. To measure mitochondrial enzyme activities, we used the Complex II Enzyme Activity Microplate Assay Kit (ab109908, Abcam) and Complex IV Rodent Enzyme Activity Microplate Assay Kit (ab109911, Abcam).

The following assays were measured from serum: aspartate and alanine aminotransferase (AST and ALT) levels were measured using AST and ALT activity assays (Sigma Aldrich, St. Louis, MO, USA). Free cholesterol and cholesteryl esters were measured using the Cholesterol Fluorometric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). LDL/VLDL levels were measured using the Cholesterol Assay Kit (Abcam, Boston, MA). Circulating triglyceride levels were measured from liver homogenates (∼100 mg) and serum using Triglyceride Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA).
Mitochondrial oxidative phosphorylation system (OXPHOS) in complexes I, II, and IV were analyzed using Complex I Enzyme Activity Colorimetric Assay Kit, Complex II Enzyme Activity Microplate Assay Kit, and the Complex IV Rodent Enzyme Activity Microplate Assay Kit (Abcam, Boston, MA, USA) using isolated mitochondria that were purified from the liver tissues using the Mitochondria Isolation Kit for Tissue (Abcam, Boston, MA, USA).
Cytokine levels were measured using Proteome Profiler Array Mouse Cytokine Array Kit Panel A (R&D Systems, Minneapolis, MN, USA) and signal density was quantified by densitometry using ImageJ software.