Ab201111

Mitochondria were extracted from PBMC using the Mitochondria Isolation Kit for Cultured Cells (Abcam, ab110170). Protein concentration of PBMC lysate was measured by BCA assay (Abcam, ab102536) and pellets were frozen at -80°C for 10 min to weaken cellular membranes then supplemented with 0.2 μL of universal nuclease (Fisher Scientific Co., PI88700) to reduce viscosity. Samples were resuspended to 5 mg/ mL in reagent A followed by homogenization. The homogenate was centrifuged at 1,000 × g for 10 min at 4°C, saving the supernatant and resuspending the pellet in reagent B. Homogenization and spin steps were repeated and the supernatants were combined and further centrifuged at 12,000 × g for 15 min at 4°C. The resulting supernatant was discarded, and the crude mitochondrial pellet dissolved in reagent C supplemented with protease inhibitor (Abcam, ab201111), aliquoted, and stored at -80°C. The crude mitochondrial protein concentration of one aliquot per sample was measured by bicinchoninic acid assay and used to correct the final activities of each sample (Abcam, ab102536).

Mitochondria were extracted from PBMC using the Mitochondria Isolation Kit for Cultured Cells (ab110170, Abcam). Lysate protein concentration from PBMC was measured by the Pierce BCA Protein Assay Kit (Thermo Scientific) and PBMC were frozen at -80°C for 10 min to weaken cellular membranes, followed by the addition of 0.2 μL of universal nuclease (PI88700, Fisher Scientific Co.). Samples were resuspended to a final protein concentration of 5 mg/ mL with reagent A, followed by homogenization and centrifugation at 1,000 × g for 10 min at 4°C. The resulting supernatant was retained and the pellet was resuspended in reagent B in the same volume used for reagent A. Homogenization and centrifugation steps were repeated. The supernatant from reagent B was combined with that kept from reagent A and centrifuged at 12,000 × g for 15 min at 4°C. This final supernatant was discarded, and the resulting crude mitochondrial pellet was dissolved in 500 μL of reagent C supplemented protease inhibitor (ab201111, Abcam), and aliquoted into 5 micro-centrifuge tubes and stored at -80°C for enzymatic analyses. The crude mitochondrial protein concentration of one aliquot per cow was measured by BCA (ab102536, Abcam). Before downstream enzymatic analysis, the protein concentration of each sample was adjusted to meet the specifications of each assay protocol.

Mitochondria were extracted from PBMC using the Mitochondria Isolation Kit for Cultured Cells (ab110170, Abcam). Lysate protein concentration from PBMC was measured by BCA assay (ab102536, Abcam) and pellets were frozen at -80°C for 10 min to weaken cellular membranes, and then supplemented with 0.2 μL of universal nuclease (PI88700, Fisher Scientific Co., Fair Lawn, NJ). Samples were resuspended to 5 mg/mL in reagent A followed by homogenization. The homogenate was centrifuged at 1,000 × g for 10 min. The resulting supernatant was saved and the pellet was resuspended in reagent B. Homogenization and spin steps were repeated once and the supernatants were combined and further centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was discarded and the resulting crude mitochondrial pellet was dissolved in reagent C supplemented with protease inhibitor (ab201111, Abcam), aliquoted, and stored at -80°C. The crude mitochondrial protein concentration of 1 aliquot per sample was measured by BCA assay and used to correct the final mitochondrial enzyme activities (ab102536, Abcam).