Bsa conjugated oleate acid palmitate acid oa pa

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.

When the cells reached 90% confluency, they were ready to be lysed ATP production analysis using an enhanced ATP Assay Kit (Beyotime, China). The RLU or CPM levels were detected using a luminometer to evaluate ATP levels. The protein concentration of lysed samples was measured using Bradford assay kit (Sigma) to calculate the average ATP yield per μg of cell protein.
The oxygen consumption rate (OCR) was determined according to the protocols described previously using a Seahorse XF-96 extracellular flux analyzer (Seahorse Bioscience, Billerica). Approximately, 4 × 10⁴ mHCs were plated in 24-well plates and treated with F12/DMEM containing 1 mM bovine serum albumen (BSA)-conjugated oleate acid/palmitate acid (OA/PA) (Sigma) and incubated for 24 h before detection. Three independent repetitions were performed. The protein concentration in each well was quantified using the BSA assay according to the manufacturer’s instructions (Thermo). The OCR was normalized to the total protein concentration in each well.