Mitochondrial complex 1 activity colorimetric assay kit

Mitochondrial respiratory chain complex I to IV (CI-IV) was detected as described before.¹⁵ (link) Mitochondria were isolated, and the levels of CI-IV and citrate synthase (CS) activity were measured separately with a Mitochondrial Complex I Activity Colorimetric Assay Kit, a Succinate Dehydrogenase Activity Colorimetric Assay Kit, a Mitochondrial Complex III Activity Assay Kit, a Cytochrome Oxidase Activity Colorimetric Assay Kit, and a Citrate Synthase Activity Colorimetric Assay Kit (all from BioVision, Milpitas, CA, USA). Each experiment was performed in three replicates and repeated four times.

In this test, PC12 cells (5 × 10⁴/mL) and C6/36 cells (1 × 10⁵/mL) were seeded in 6-well plates for 2 mL each well and cultivated for 24 h, and then eugenol was added to the medium to the final concentrations of 25 and 50 μg/mL for the incubation. Rotenone (50 μg/mL) was used as control. Each group was set to 6 multiple well. After treatment for 48 h, PC12 cells and C6/36 cells were collected. Then, the cells were harvested and combined to isolate the mitochondria; 1% DMSO was applied to an untreated group. According to the above methods, mitochondria (protein concentration 0.5 mg/mL) were isolated from PC12 and C6/36 cells, and 1 μg of mitochondria was used to determine the respective complex I activity using a Mitochondrial Complex I Activity Colorimetric Assay Kit (Biovision, U.S.A.). Five replicates were performed.

Activity of mitochondrial complex I was evaluated using the Mitochondrial Complex I Activity Colorimetric Assay Kit (BioVision). Mitochondria were isolated from cells of interest, and a 5-μg sample was reacted with complex I assay buffer, decylubiquinone, and complex I dye, with or without rotenone addition. OD₆₀₀ was measured in kinetic mode at 30-s intervals for up to 5 min at room temperature. Activity of mitochondrial complex I was calculated based on a standard curve.

The inhibition of complex I activity in the electron transport chain was measured using a Mitochondrial Complex I Activity Colorimetric Assay Kit (Biovision, U.S.A.) following the manufacturer's protocol. Eugenol at final concentrations of 2, 10 and 20 ng/mL was used as an inhibitor in this test, rotenone (2 ng/mL) was used as a positive control, and 10% DMSO was used as a negative control. The activity was determined by measuring the decrease in absorbance/min at room temperature at 600 nm with an enzyme-linked immunosorbent assay microplate reader (Multiskan MK3, Thermo Scientific, U.S.A.). Absorbance was recorded every 1 min for 5 min. Three replicates were performed for each group.
To study the effect of succinate on the inhibition of complex I by eugenol, a polarographic study was performed. Eugenol at a final concentration of 20 ng/mL was used as an inhibitor in this test, succinate (2–20 ng/mL) was added independently or concurrently with eugenol, and 10% DMSO was used as a negative control. The activity was determined by measuring the decrease in absorbance in OD/min at room temperature at 600 nm. Absorbance was recorded for 5 min, and inhibition rates of chemicals against complex I activity were calculated. Three replicates were performed for each group.