Extracellular oxygen consumption assay kit

Hepatocytes were isolated and cultured as described previously (Caldez et al., 2018 (link)). β-oxidation capabilities of isolated hepatocytes were then measured using the FFA Assay Kit (ab217602, Abcam) in combination with the Extracellular Oxygen Consumption Assay Kit (ab197243, Abcam) following manufacturer’s protocol. Signals were read using a TECAN Safire microplate reader with parameters as indicated in the assay protocol.

After 2 days of differentiation, adipocytes in a 96-well plate were transfected with overexpression vectors or siRNAs. After 6 days of transfection, the oxygen consumption rate (OCR) was measured using the Extracellular Oxygen Consumption Assay Kit (Abcam, Cambridge, UK), guided by the manufacturer indications.

The oxygen consumption assay was performed using Abcam’s extracellular oxygen consumption assay kit (ab197243). Assay was performed as per the manufacturer’s suggested protocol. Exponentially growing bacterial cultures were diluted to an OD₆₀₀ of ∼0.1, and 100 μl of the diluted cultures was added to a flat and transparent bottom 96-well sterile half-area cell culture plate. Dye was added as suggested in the manufacturer’s protocol, and wells were sealed with mineral oil. The time-resolved fluorescence was recorded in a kinetic cycle with 2 min interval with a 30-μs excitation-emission time lag and a 100-μs integration time using a Tecan infinite 200Pro. Plates were incubated at 37°C in a shaking mode (2-mm orbital amplitude). The OD₆₀₀ was recorded in parallel to normalize for the cell number. Three independent replicates were used for every condition.

Cell extracellular oxygen consumption and extracellular acidification were measured using Extracellular Oxygen Consumption Assay Kit (Abcam, Cambridge, UK) and Glycolysis Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Cells were treated with MCL1 inhibitor S63845 and metformin for 24 h and 72 h before measurements. Cells were harvested, washed, and seeded at 5 × 10⁵ viable cells/well in a 96-well plate, detection reagents were added and measurements performed in a plate reader. The aforementioned assays were validated using glucose oxidase as a positive control (Sigma-Aldrich, St. Louis, MO, USA). Cellular ATP concentration was evaluated using Luminescent ATP Detection Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. ATP standards provided in the kit were used in order to test the validity of the assay. Cells were seeded at 7.5 × 10⁴ viable cells/well in a white 96-well plate, detergent solution was added and incubated for 5 min to lyse cells and stabilize ATP, then substrate solution was added and the plate was stored in the dark until analysis of luminescence. For cell metabolic activity measurements the Thermo Scientific Varioskan^® plate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used.