Bsa conjugated palmitate

Cellular OCR was used to evaluate the capacity of fatty acid oxidation in real time using the Seahorse XF96 Extracellular Flux Analyzer. 2×10⁴ SMMC-7721 vector or HBx cells per well were seeded in triplicates in an XF96 well culture plate. Then the growth medium was replaced with substrate-limited DMEM supplemented with 0.5 mM glucose, 1 mM glutamine, 0.5 mM carnitine, 1% FBS and maintained overnight in 37°C. On the day of assay, the FAO assay KHB buffer (110 mM NaCl, 4.7 mM KCl, 2 mM MgSO₄, 1.2 mM Na₂HPO₄) supplemented with 2.5 mM glucose, 0.5 mM carnitine and 5mM HEPES was exchanged and adjusted to pH 7.4 in 37°C incubator. To examine free fatty acid oxidation, BSA conjugated Palmitate (Seahorse Bioscience) was added to a final concentration of 50 μM. Basal OCR of cells treated with Palmitate-BSA or BSA vehicle alone were measured.

RPE were cultured on 96 well XF Microplates (Agilent Technologies Inc.; La Jolla, CA)) and maintained as described above. Prior to analysis human RPE were transferred to assay media (modified Ringer’s solution lacking sodium bicarbonate). For glucose metabolism assays media was supplemented with 12 mM glucose, 2 mM pyruvate, 2 mM glutamine, and 10 mM HEPES, pH 7.4. For fatty acid oxidation (FAO) assays the media contained 2.5 mM glucose, 0.5 mM carnitine and 10 mM HEPES, pH 7.4. Oxygen consumption rates (OCR) were measured using a XF^e96 device (Agilent Technologies Inc.)); the following concentrations of drugs were used: 2 µM oligomycin, 0.5 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 1 µM antimycin A, and 1 µM rotenone. For measuring fatty acid oxidation higher concentrations of drugs were used: 2 µM oligomycin, 1 µM FCCP, 4 µM antimycin A, and 2 µM rotenone. For FAO, etomoxir (40 µM/well) was added at least 30 min prior to analysis. BSA and BSA-conjugated palmitate (Agilent Technologies Inc.) were added to the cells immediately prior to reading. Basal OCR was calculated by subtracting the OCR in the presence of antimycin A and rotenone from that of the initial OCR prior to addition of drugs. Maximal OCR was calculated by subtracting OCR in the presence of antimycin A and rotenone from the OCR in the presence of FCCP.

The FAO rate analyses were performed using the Agilent Seahorse XF Cell Mito Stress Test Kit (Agilent, 103015‐100) as previously described.^[89 (link)
^] Briefly, 15 000 cells per well were seeded in a 96‐well XF cell culture microplate in the growth medium overnight at 37 °C in 5% CO₂. Then, cells were starved by incubating with substrate‐limited DMEM supplemented with 0.5 mm glucose, 1 mm glutamate, 0.5 mm carnitine, and 1% FBS. After 24‐h starvation, the medium was replaced by FAO assay medium (111 mm NaCl, 4.7 mm KCl, 1.25 mm CaCl₂, 2 mm MgSO₄, 1.2 mm NaH₂PO₄ supplemented with 2.5 mm glucose, 0.5 mm carnitine, and 5 mm HEPES, pH 7.4). Finally, BSA‐conjugated palmitate (Agilent, 102720‐100) was added to a final concentration of 50 mm, followed by sequential addition of 1.5 µm oligomycin, 1.0 µm FCCP, 0.5 µm rotenone, and 0.5 µm antimycin A. Data were analyzed by the Seahorse XF Cell Mito Stress Test Report Generator package.