Cytochrome oxidase activity colorimetric assay kit

Mitochondria were isolated from HMLE/LACZ and /EGFL9 cell lines using the Mitochondrial Isolation Kit for Cultured Cells (Thermo Fisher Scientifics). The Cytochrome Oxidase Activity Colorimetric Assay Kit from Biovision was used to measure cytochrome oxidase activity. In all, 2.5 µg of mitochondria were plated in a 96-well plate with a mixture of reduced cytochrome oxidase and buffer per manufacturer instructions. Then, the OD at 550 nm was measured and the maximum linear decay was used to calculate activity (units/mg). The difference between groups was evaluated for statistical significance by a two-sample T-test. The assay was performed in triplicate, with two blank wells containing only the reduced cytochrome c/buffer mixture as a negative control.

HMEECs were incubated with 0, 1.5, and 2 mM FA for 4 h and 0, 0.1, and 0.2 μM FA for 24 h. Apoptosis was then analyzed using a cytochrome oxidase activity colorimetric assay kit (BioVision, Milpitas, CA, USA) after cell lysis. The change in absorbance at 550 nm via the oxidation of reduced cytochrome c, measured in six replicate samples, served as an indicator of cytochrome oxidase activity.

SCs were stained using a Cell Navigator Mitochondrion Staining Kit (Green Fluorescence; AAT Bioquest, Inc., Sunnyvale, CA) at 37 °C in a 5% CO₂ incubator for 1 h. The cells were then photographed using a fluorescence microscope at 485 nm excitation/525 nm emission. Fluorescence intensities were analyzed using ImageJ.
Mitochondria were isolated and purified from SCs using a Qproteome Mitochondria Isolation Kit (QIAGEN, Valencia, CA), according to the manufacturer’s instructions. The NADH level and enzymatic activities of cytochrome c oxidase and ATP synthase in SCs were measured using an NAD + /NADH Quantitation Colorimetric Kit (BioVision), Cytochrome Oxidase Activity Colorimetric Assay Kit (BioVision), and ATP Synthase Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany), according to the manufacturers’ instructions. Optical densities were measured using the GloMax Discover Multimode Detection System.
SC protein level was measured using the Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich) and adjusted to equal protein concentrations using PBS. ATP concentrations in SCs were assayed using a kit from Invitrogen (Thermo Fisher Scientific), following the manufacturer’s procedures. The light unit values of ATP level were determined using a luminometer (Sirius L Tube Luminometer, Titertek Berthold, Germany). Unit of ATP (nmole/mg protein) was expressed.

Cytochrome oxidase complex IV assay was performed using a commercially available cytochrome oxidase activity colorimetric assay kit according to a manufacturer's instruction (BioVision) after primary neonatal rat cardiomyocytes were treated as specified, and mitochondria isolated from them, as described above. The activity was then normalized to citrate synthase activity, which was assayed using a commercially available kit (ScienCell) according to the manufacturer's instruction after mitochondrial isolation.

Cytochrome c oxidase activity was measured using the cytochrome oxidase activity colorimetric assay kit (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer’s instructions. Briefly, the cells were harvested in a 10 cm culture dish at 70% cell density. The quantification of protein was measured by Bicinchoninic acid (BCA) assay. The assay mixture containing 20 μL reduced cytochrome C, 100 μL assay buffer, and 150 μg cell lysate was pipetted into the wells of 96 well plates. The kinetic enzyme assay was measured at an absorbance of 550 nm with VERSA maxTM to obtain the variation of colorimetric value. The kinetic protocol is that the interval time is 1 min at 25 °C by 25 min. The rate of the reaction is relative to sh-control samples. The rate was calculated in linear rage. All experiments were performed in duplicate.

TCA‐8113 and CAL‐27 cells were obtained from the West China College of Stomatology of Sichuan University (Sichuan, China). RPMI 1640 medium, nobiletin, and Sp‐cAMP were purchased from Sigma. Fetal bovine serum was purchased from GIBCO. Cell Counting Kit‐8 (CCK8) was purchased from MCE (HY‐K0301). Anti‐PCNA (ab29), anti‐cyclin D1 (ab134175), anti‐GAPDH (ab8245), anti‐PKA (ab75993), anti‐phosphorylated‐PKA (ab32390), anti‐CREB antibody (ab32515), and anti‐phosphorylated‐CREB (ab32096) antibodies, Propidium Iodide Flow Cytometry Kit (ab139418), Glucose Uptake Assay Kit (ab136955), L‐Lactate Assay Kit (ab65331), Pyruvate Assay Kit (ab65342), and pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902) were purchased from Abcam. Cytochrome Oxidase Activity Colorimetric Assay Kit was purchased from BioVision (K287). MitoTracker® Green FM KIT was purchased from Cell Signaling Technology (9074S).