Firefly luciferase based atp assay kit

The ATP levels of liver tissues from mice were measured using a firefly luciferase-based ATP assay kit (Beyotime, Shanghai, China), according to the manufacturer’s instructions. After the indicated treatments, the liver tissues were homogenated and centrifuged at 12,000×g for 5 min. The supernatants (100 μL) were mixed with 100 μL of ATP detection solution at a working dilution in a white 96-well plate. Standard curves were also generated, and the protein concentration of each treatment group was determined using a Bradford protein assay. The total ATP levels were expressed as nmol/mg protein. This experiment was repeated three times.

The transmembrane potential was determined with the MitoProbe™ DiOC₂(3) Assay Kit (Invitrogen™), and the cellular ATP levels were measured using a firefly luciferase-based ATP assay kit (Beyotime, Shanghai, China), according to the manufacturers’ protocols.

ATP levels were measured using a firefly luciferase based ATP assay kit (Beyotime, Shanghai, China), in accordance with the manufacturer's instructions. Briefly, lysed tissue suspension was centrifuged at 12,000 × g for 5 min. In a 96-well plate, 20 μL of supernatant was mixed with 100 μL ATP detection working dilution. Luminance (in relative light units, RLU) was measured by a monochromator microplate reader (Safire II, Tecan, Switzerland). Protein concentration was determined by the BCA Protein Assay kit (Solarbio). Total ATP levels were expressed as nmol/mg protein.

Cellular ATP levels were measured with a firefly luciferase‐based ATP assay kit (Beyotime). Briefly, primary hepatocytes, in triplicate sets, were lysed and centrifuged at 12,000 × g for 5 min, and 100 μl of supernatant was mixed with 100 μl of ATP detection solution. Total ATP levels are expressed as nmol/mg protein. The intact cellular oxygen consumption rate (OCR) was measured using a Seahorse XF‐96 extracellular flux analyzer (Seahorse Bioscience) as described previously (Gaude et al., 2018; Xie et al., 2016). The results were obtained by performing in triplicate in 24‐well plates, 4 × 10⁴ hepatocytes each, the protein concentration in each well was measured by BCA assay according to the manufacturer's instructions (Thermo), and the protein concentrations are around 0.2–0.3 μg/ml. The OCR value was normalized to the total protein level in each well. Hepatocytes were treated with DMEM/F12 containing 1 mM BSA‐conjugated oleate acid/palmitate acid (OA/PA) (Sigma‐Aldrich) and incubated for 24 hours before the measurement.