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MS, MultiSensitive Phosphor Screens, Medium

MS, MultiSensitive Phosphor Screens, Medium

Company: PerkinElmer
Catalog#: 7001723
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Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes
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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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