Complex 1 enzyme activity dipstick assay kit

Complex I activity in whole cells was measured using the Complex I Enzyme Activity Dipstick Assay Kit (ab109720, ABCAM, Cambridge, MA, USA) according to manufacturer’s instructions. Three biological replicates were used per measurement. Results are expressed as enzyme activity respect to control. The signal intensity was analyzed by a Molecular Imager ChemiDoc XRS + System (Bio-Rad Laboratories Inc., USA).

Complex I activity in whole cells was measured using the Complex I Enzyme Activity Dipstick Assay Kit (ab109720, ABCAM, Cambridge, MA, USA) according to manufacturer’s instructions. Three biological replicates were used per measurement. Results are expressed as enzyme activity respect to control. The signal intensity was analyzed by a Molecular Imager ChemiDoc XRS + System (Bio-Rad Laboratories Inc., USA).

Complex I and IV activities were measured in INS-1 832/13 cell extracts using Complex I Enzyme Activity Dipstick Assay Kit (Abcam, cat# ab109720) and Complex IV Rodent Enzyme Microplate Assay Kit (Abcam, cat# ab109911) following the manufacturer’s protocols. Whole-cell extracts (25 µg for Complex I assay and 50 µg for Complex IV assay) were used. Aconitase activity was measured in INS-1 832/13 cells enriched mitochondrial and cytosolic extracts (Mitochondria Isolation Kit for Cultured Cells, ThermoFisher, cat# 89874) using the Aconitase Enzyme Activity Microplate Assay Kit assay (Abcam, cat# 109712). Cell lysates (n = 4 independent biological experiments) were stored at −80 °C and then assayed together. For measurement of intracellular ATP, INS-1 832/13 cells were preincubated in HBSS (secretion buffer) with 2.5 mM glucose for 30 min at 37 °C. Cells were then incubated with 5 mM glucose or with 16.7 mM glucose for 1 h. ATP content was measured in triplicate samples using the ATP Determination Kit (Molecular Probes, cat# A22066) and normalized to total cellular protein. For enzyme activity and ATP assays, cells were treated with FAC (10 µg/mL) for 18 h before performing the assay.

Mitochondrial Complex I activity in the dorsal cortex of the brain was estimated using the Complex I enzyme activity dipstick assay kit (Abcam). Dorsal cortex was isolated from freshly dissected coronal brain slices and stored frozen at -80°C until processing. Tissue lysates were obtained by mechanical homogenization (Dounce homogenizer) in 200 μL of the extraction buffer supplemented with protease inhibitor and phosphatase inhibitor cocktails (Sigma). The homogenates were incubated on ice for 20 min and then centrifuged at 16000 × g for 30 min. The supernatant was collected for the enzymatic assay and protein concentration was determined by Bradford assay (BioRad). To measure MCI activity, 5 μg of each protein extract was used. Images from the developed dipsticks were acquired (ImageQuant LAS 4000 mini, GE Healthcare) and signal intensity was quantified using the ImageQuant TL software (GE Healthcare). Interpolation from a standard curve was performed.