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9 protocols using enzymatic kit

1

Lipid Extraction and Quantification

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Tissues were sonicated twice for 10 s on ice in lysis buffer (100 mM potassium phosphate (Carl Roth, Karlsruhe, Germany), 250 mM sucrose (Carl Roth, Karlsruhe, Germany), 1 mM EDTA (ThermoFisher Scientific, Waltham, MA, USA), pH 7) and the protein amount was determined after centrifugation as described above. Lipids were extracted from 1 mg protein following Folch’s method [48 (link)]. TG, TC, and FC concentrations were determined using enzymatic kits following the manufacturer’s guidelines (DiaSys, Holzheim, Germany). CE concentrations were calculated by subtraction of FC from TC values. All lipid concentrations were normalized to protein content.
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2

Plasma Lipid and Metabolite Analysis

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Blood was collected by facial vein puncture and plasma was prepared within 20 min. TG, total cholesterol (TC), and free cholesterol (FC) concentrations were determined in plasma from mice using enzymatic kits (DiaSys, Holzheim, Germany). Plasma free glycerol (FG) and free fatty acid (FFA) concentrations were determined using Free Glycerol Reagent (Sigma-Aldrich, St. Louis, MO) and NEFA-HR kit (Wako Life Sciences, Mountain View, CA), respectively. Lipoprotein fractions were separated from 200 μl pooled plasma from each group using fast protein liquid chromatography as previously described [24 (link)]. Creatine phosphokinase activity in plasma samples was measured using a Spotchem EZ analyzer and test strips (A. Menarini GmbH, Vienna, Austria).
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3

Liver Lipid Extraction and Quantification

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Half of the largest liver lobe was lysed on ice in lysis buffer by 6 × 10 s sonication. Lysates were centrifuged for 10 min at 20,000 g and 4°C, and protein concentrations in the supernatants were measured using the DC™ Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Lipids were extracted from the lysates containing 1 mg of protein by the Folch method. TG and TC concentrations were determined using enzymatic kits (DiaSys, Holzheim, Germany) according to the manufacturer's guidelines.
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4

Lipid Quantification in Cell Lysates

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Cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton® X-100, 0.1% SDS, 50 mM Tris, 0.5% Na-deoxycholate, pH 8) supplemented with a protease inhibitor cocktail (P8340, 1:1,000; Sigma–Aldrich, St. Louis, MO)) by 2 × 10 s sonication on ice. Protein concentration was determined by a Lowry assay (Bio-Rad Laboratories, Hercules, CA). Concentrations of TG, TC, and free cholesterol (FC) were measured with enzymatic kits (DiaSys, Holzheim, Germany), whereas CE concentrations were calculated by subtracting FC from TC.
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5

Serum and HDL Lipid Analysis

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Total serum and HDL lipids were measured using enzymatic kits from DiaSys Diagnostic Systems GmbH (Holzheim, BY, Germany). Total unesterified cholesterol in serum samples was determined by an enzymatic kit from Sigma Aldrich (St. Louis, MO, USA). Total HDL protein content was measured using BCA kit from Thermo Fischer Scientific Inc. (Waltham, MA, USA).
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6

Biochemical Assays of Metabolic Markers

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Serum glucose was assayed using enzymatic kits (DiaSys Diagnostic Systems GmbH, Holzheim, Germany). The serum free fatty acid concentration was assayed with a Roche FFA kit (Roche Applied Science). Serum glycerol was measured using the Free Glycerol Reagent (Sigma). Serum insulin, leptin and adiponectin concentrations were measured by radioimmunoassay (Millipore).
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7

Plasma Cholesterol Profiling in Mice

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Blood samples were collected from mice. Plasma was separated by centrifugation, and cholesterol levels were measured using an enzymatic kit (catalog no.: 113009910026; Diasys Diagnostic Systems, Holzheim, Germany) with Cholesterol FS standard (catalog no.: 113009910030; Diasys Diagnostic Systems) for the calibration curve. Lipoprotein cholesterol distribution was measured by fast performance liquid chromatography using a system containing a PU-4180 pump with a linear degasser and UV-4075 UV/VIS detectors (Jasco, Tokyo, Japan). Pooled plasma samples (n = 15–16 mice per pool) were injected onto a Superose 6 Increase 10/300 GL column (GE Healthcare, Hoevelaken, The Netherlands) and eluted at a constant flow rate of 0.31 ml/min in PBS (pH 7.4). Cholesterol was measured in line by addition of cholesterol reagent at a constant flow rate of 0.1 ml/min using an additional PU-4080i infusion pump (Jasco, Tokyo, Japan). Data acquisition and analysis were performed using ChromNav software (version 1.0; Jasco, Tokyo, Japan).
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8

Extraction and Quantification of Kidney Triglycerides

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Total lipids were extracted from kidney tissue according to Folch et al. [42 (link)]. The organic layer was dried under nitrogen gas and solubilized in isopropanol/Triton X-100 (10%) and then assayed for triglyceride concentration using an enzymatic kit (DiaSys). Data were expressed as the amount of triglycerides per gram of wet weight.
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9

Placental Lipid Extraction and Triglyceride Assay

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Placental villous tissue (50 mg) was homogenized in 1 ml acetone and total lipids were extracted by overnight incubation on a shaking platform. Samples were centrifuged at 16 000 g for 15 min, and 5 μl acetone extract was used to assay the TG concentration by an enzymatic kit (Diasys Diagnostic Systems) following the manufacturer's instructions.
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