The β-oxidation rate of free fatty acid was measured as previously reported,63 (link) with some modifications. HepG2 cells were cultured in DMEM medium on 6-well plate to a density of 1×106 cells per well. For the assay, add to each well 1ml Krebs-Ringer phosphate buffer containing fatty acid-free BSA (20 mg·ml−1), glucose (2.5 mM), oleic acid (0.4 mM, Sigma), and 1-14C oleic acid (1.0 μCi·ml−1, GE Healthcare). To start the treatment, NEN (1.0 μM) or vehicle were added into each well, which was then covered by a piece of square filter paper soaked with hyamine hydroxide (Perkin Elmer). After incubation for 3 h, 0.5 ml of 4 M H2SO4 was added to each well and then the wells were sealed again for additional 30 min before the filter papers were carefully removed. The 14CO2 trapped on each filter paper was measured by a scintillation counter (Beckman LS 5000 TD Liquid Scintillation System).
1 14c oleic acid
Manufactured by GE Healthcare
1-14C oleic acid is a radiolabeled fatty acid used in research applications. It contains a carbon-14 (14C) label at the first carbon position of the oleic acid molecule. This product can be used as a tracer compound to study lipid metabolism and transport processes.
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2 protocols using 1 14c oleic acid
1
Measuring Fatty Acid β-Oxidation in HepG2 Cells
2
Measuring Fatty Acid β-Oxidation in HepG2 Cells
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The β-oxidation rate of free fatty acid was measured as previously reported,63 (link) with some modifications. HepG2 cells were cultured in DMEM medium on 6-well plate to a density of 1×106 cells per well. For the assay, add to each well 1ml Krebs-Ringer phosphate buffer containing fatty acid-free BSA (20 mg·ml−1), glucose (2.5 mM), oleic acid (0.4 mM, Sigma), and 1-14C oleic acid (1.0 μCi·ml−1, GE Healthcare). To start the treatment, NEN (1.0 μM) or vehicle were added into each well, which was then covered by a piece of square filter paper soaked with hyamine hydroxide (Perkin Elmer). After incubation for 3 h, 0.5 ml of 4 M H2SO4 was added to each well and then the wells were sealed again for additional 30 min before the filter papers were carefully removed. The 14CO2 trapped on each filter paper was measured by a scintillation counter (Beckman LS 5000 TD Liquid Scintillation System).
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