Spheroid cultures were exposed to a lipogenic cocktail of FFA, as previously described23 (link), with minor modifications. Unsaturated oleic acid (Sigma-Aldrich) and saturated palmitic acid (Sigma-Aldrich), solubilized in ethanol, were conjugated to 10% bovine serum albumin (BSA; Sigma-Aldrich) at a 1:5 molar ratio for 2 h at 40 °C. The FFA were combined in a 1:1 ratio. Spheroids were treated with 480 µM FFA from day 7 to day 14, with medium changed every 2–3 days.
Saturated palmitic acid
Manufactured by Merck Group
Sourced in United States
Saturated palmitic acid is a laboratory reagent used in various scientific applications. It is a long-chain fatty acid with the chemical formula CH3(CH2)14COOH. Palmitic acid is a white, odorless, and waxy solid at room temperature. It is commonly used as a reference standard, ingredient in culture media, and in the synthesis of other compounds.
Automatically generated - may contain errors
Lab products found in correlation
Unsaturated oleic acid, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
L glutamine, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Hepatozyme sfm medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
4 protocols using saturated palmitic acid
1
Lipogenic Cocktail Exposure in Spheroids
2
Lipogenic Exposure of Spheroid Cultures
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Spheroid monocultures and co-cultures were exposed to lipogenic substances, as previously described (25), with minor modifications. Unsaturated oleic acid and saturated palmitic acid (Sigma Aldrich, MO, USA), solubilized in ethanol, were conjugated to 10% bovine serum albumin at a 1:5 molar ratio for 2 h at 40 °C. The free fatty acids (FFA) were combined in a 1:1 ratio and spheroids were exposed to 480 µM from day 5 to 14 with medium exchanged every 2 to 3 days.
3
Hepatic Spheroid Lipid Metabolism
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Exposure and treatment with lipogenic substrates started at day 7 after seeding of PHH when the spheroids had been formed, which hereafter is regarded to as day 0. Spheroids were exposed to either physiological medium (100 pM insulin, 5.5 mM glucose) or lipogenic media containing higher levels of insulin supplemented with free fatty acids and monosaccharides (glucose and fructose), see Results section. A combination (1:1 ratio) of the saturated palmitic acid (Sigma-Aldrich) and unsaturated oleic acid (Sigma-Aldrich) was used to mimic human plasma concentrations of free fatty acids. In order to facilitate free fatty acid uptake to the hepatic spheroids, the free fatty acids were bound to 10% bovine serum albumin (Sigma-Aldrich) at a molar ratio of 1:5 for 2 hours at 40 °C. Spheroids were exposed every 48–72 hours with the indicated substrates.
4
Isolation and Stimulation of Primary Murine Hepatocytes
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Hepatocytes were isolated from livers of 9∓13-week-old WT and Plin5−/− male mice using the collagenase method [17 (link)]. Hepatocytes were cultured on collagen-coated dishes (3 × 104 cells/well) in a Hepatozyme-SFM medium (Gibco, St. Louis, MO, USA). After cell adherence, the medium was changed to DMEM supplemented with 10% fetal calf serum (FCS), 1% pyruvate, 1% Penicillin/Streptomycin solution and 4 mM L-Glutamine (all from Sigma-Aldrich, Taufkirchen, Germany). The day after, the cells were starved for 3 h in DMEM containing 0.5% FCS and then stimulated with fatty acids or lipopolysaccharide (LPS) in a medium containing 0.2% FCS. After the respective treatments, the cells and supernatants were harvested for RNA and/or protein extraction as described elsewhere [16 (link)]. For stimulation experiments, primary hepatocytes from WT and Plin5−/− mice cultured overnight (described above) were stimulated after starvation by the addition of 0.5 mM unsaturated oleic acid or saturated palmitic acid (both from Sigma-Aldrich). Untreated or vehicle-stimulated cells were used as the control. For LPS stimulation, primary hepatocytes were treated after the 3-h starvation period with 1 μg/mL LPS (Sigma-Aldrich) for 3 or 24 h.
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