Cytochrome c oxidase assay kit

Cytochrome C oxidase activity was measured with Cytochrome C Oxidase Assay Kit (Millipore-Sigma) in accordance with manufacturer's instructions.

The CCO activity was determined in mitochondria-enriched sperm fractions, as described in Mclean et al. (1993) (link). Briefly, 1-mL sperm aliquots, previously irradiated with red light, were centrifuged at 1,000 × g and 17°C for 30 s. The resulting pellets were immediately plunged into liquid N₂ and stored for 3 weeks. Pellets were resuspended in 500 μL ice-cold PBS and sonicated (10 kHz, 20 pulses; Bandelin Sonopuls HD 2070). Thereafter, 500 μL Percoll^® (concentration: 1.055 mg/mL in PBS) at 4°C was placed onto each sperm homogenate. Samples were centrifuged at 3,000 × g and 10°C for 45 min and the mitochondria-enriched fraction was carefully harvested with a micropipette and transferred into a new 1.5 mL tube. Samples were again centrifuged at 12,000 × g and 20°C for 2 min and the resulting pellets were resuspended in 100 μL PBS at 20°C. These mitochondria-enriched suspensions were split into two separate aliquots. The first one was used to determine CCO activity using a commercial kit (Cytochrome C Oxidase Assay Kit; Sigma-Aldrich; catalog number CYTOCOX1). Enzyme activity was normalized against the total protein content. Therefore, the other aliquot (10 μL) was used to determine total protein content through a commercial kit (Bio-Rad laboratories) based on the Bradford method (Bradford, 1976 (link)).

Mitochondrial cytochrome c oxidase activity was measured in non‐Tg (n = 6) and Mpst‐Tg mice (n = 6) (both age of 6 weeks). Fresh mitochondria were isolated from cerebral hemisphere by using Mitochondria Isolation Kit for Tissue and Cultured Cells (Biochain, Newark, CA, USA) and collected pellets were stored at −80°C until use. At the timing of measurement, pellets were re‐suspended in the storage buffer [10 mM HEPES (pH 7.1–7.5), 250 mM sucrose, 1 mM ATP, 80 μΜ ADP, 5 mM sodium succinate, and 2 mM K₂HPO₄]. The activity was determined by Cytochrome c Oxidase Assay Kit (Sigma). The assay was performed using Ultrospec 3300 pro (GE Healthcare). Results were represented in units per mg of protein defined by Micro BCA Protein Assay Kit (ThermoFisher).

Total hind limb muscle, liver, heart, or brain mitochondria were prepared by differential centrifugation and purification on a Percoll gradient as we have described in the past [10 (link)]. Mitochondrial integrity was assessed by cytochrome C release using a commercial kit (Cytochrome C Oxidase Assay Kit, Sigma-Aldrich, St. Louis) indicating a mean of 96% intact mitochondria over three assays, well within an acceptable range compared to mitochondrial preparations from several sources [11 (link)].

Plants were grown hydroponically with or without 250 nM CuSO₄ for four weeks (in case of rosette leaves) or seven to eight weeks (in case of floral buds). Mitochondria were extracted according to the procedure described in (Keech et al., 2005). This experiment was done three independent times for each tissue, each with three independent biological replicates, with all experiments showing similar results. Cytochrome c oxidase activity was measured using Cytochrome c Oxidase Assay Kit (Sigma‐Aldrich, catalog number CYTOCOX1) according to the manufacturer's instructions.