| Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes
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Author:
Date:
2016-11-05
[Abstract] Metabolic flux analyses are needed to provide insights into metabolic regulation that occurs in cells. The current protocol describes fast and reproducible methods for determining glycolysis and de novo lipogenesis of hepatocytes. Primary culture of hepatocytes is an ‘in vitro’ model useful to study liver glucose and lipid metabolism (Denechaud et al., 2016). The protocol is divided in 2 parts. Part I: Glycolysis experiment is assessed using the Seahorse extracellular flux (XF) analyser. Glycolysis is determined via the measurement of the extracellular acidification rate (ECAR) of the media, which come predominately from the cellular excretion of lactic acid after the conversion of glucose in pyruvate. Part II: De novo lipogenesis experiment determines ...
[摘要] 需要代谢通量分析来提供细胞中发生的代谢调节的见解。目前的协议描述了确定糖酵解和肝细胞脂肪生成的快速和可重复的方法。肝细胞的原代培养是用于研究肝葡萄糖和脂质代谢的"体外"模型（Denechaud等人，2016）。协议分为2部分。第I部分：使用海马细胞外通量（XF）分析仪评估糖酵解实验。糖酵解通过测量培养基的细胞外酸化速率（ECAR）来确定，所述培养基主要来自丙酮酸中葡萄糖转化后乳酸的细胞排泄。第II部分：新生脂肪生成实验测定来自乙酸盐C 14+前体的甘油三酯（TG）中的放射性C 14+掺入。在2小时后，向培养基中补加乙酸盐，提取脂质，并在新合成的TG标记的定量之前通过TLC（薄层色谱）分离。
[背景] 有不同的方法来评估葡萄糖和脂质代谢：代谢物定量，酶活性和代谢组学...我们的协议聚焦于活细胞的代谢通量分析并且不需要代谢组学设施。海马细胞外通量（XF）分析仪，现在存在于很多机构，是通过确定培养基pH值间接测量活细胞间接糖酵解的强大工具。脂肪生成协议不需要大投资，是高度可重复的。也可以使用氚化水确定，其通过脂肪酸合酶引入新鲜合成的脂质中。
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| Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient
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Author:
Date:
2016-10-20
[Abstract] Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This article describes the protocols for 1) extraction of total lipids from eukaryotic cells using the method developed by Bligh and Dyer (1959), 2) the quantification of glycerophospholipids by gas chromatography after methanolysis, followed by 3) the formation of LUVs by extrusion, 4) protein-lipid binding assay, 5) analysis of the incubation product by transmission electron microscopy (TEM) and by flotation across a discontinuous sucrose gradient and ...
[摘要] 解剖在给定类型的细胞中蛋白质和膜之间建立的相互作用不是一个容易的任务。使用大单层囊泡（LUV）的无细胞系统来分析这些相互作用可以帮助破译这些相互作用和识别由与这些LUV孵育的蛋白质诱导的潜在的膜变形。本文介绍了1）使用由Bligh和Dyer（1959）开发的方法从真核细胞中提取总脂质，2）在甲醇分解后通过气相色谱法定量甘油磷脂，然后3）通过挤出形成LUV的方案， 4）蛋白质 - 脂质结合测定，5）通过透射电子显微镜（TEM）和通过不连续蔗糖梯度浮选分析孵育产物，最后，6）通过免疫印迹分析蛋白质并通过碘素熏蒸显示甘油磷脂。
[背景] 包含巨单层囊泡（GUV;由单个磷脂双层组成，直径大于1μm）或脂质体孵育的无细胞系统与重组蛋白可能有助于了解这些相互作用。根据它们的直径和层数，脂质体被分为小的单层囊泡（SUV;由单个磷脂双层构成的囊泡，直径在20和100nm之间），大的单层囊泡（LUV;由单个双层磷脂，并且直径在100和400nm之间），大多层囊泡（MLV;由多个磷脂双层构成且直径在200nm和3μm之间的囊泡）和多泡囊泡（MVV）;由囊泡组成的大囊泡单个双层磷脂，并含有几个较小的囊泡，每个囊泡由单个双层磷脂组成）。 ...
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