Mitochondrial Dehydrogenase Activity Assay

The harvested cells were added to metabolites and antibiotic and incubated at 30°C for 6 h. After incubation, cells were collected and re-suspended in sterile saline to OD₆₀₀ = 1.0. Samples with 1 mL were collected by centrifugation at 8,000 rpm for 5 min. Pellets were re-suspended in PBS and broke down by sonication for 2 min at a 200 W power setting on ice, and then centrifuged at 12,000 rpm for 10 min to remove insoluble material. Supernatants containing 400 µg total proteins were transferred to pyruvate dehydrogenase (PDH) reaction mix (0.5 mM MTT, 1 mM MgCl₂, 6.5 mM PMS, 0.2 mM TPP, 2 mM sodium pyruvate, 50 mM PBS), ketoglutarate dehydrogenase (KGDH) reaction mix (0.5 mM MTT, 1 mM MgCl₂, 6.5 mM PMS, 0.2 mM TPP, 50 mM alpha-ketoglutaric acid potassium salt, 50 mM PBS), or succinate dehydrogenase (SDH) reaction mix (0.5 mM MTT, 13 mM PMS, 5 mM sodium succinate, 50 mM PBS), to a final volume of 200 µL in 96-well plate. Subsequently, the plate was incubated at 37°C for 5 min for SDH/PDH/OGD assays, and then measured at 566 nm for colorimetric readings. The plate was protected from light during the incubation. Experiments were repeated at least in three independent biological replicates.