二十碳细胞分析法——一种基于免疫荧光的检测方法，用于在细胞内定位新合成的类二十烷酸脂质介质。
EicosaCell - an immunofluorescent-based assay to localize newly synthesized eicosanoid lipid mediators at intracellular sites.
作者信息
Bandeira-Melo Christianne, Weller Peter F, Bozza Patricia T
机构信息
Laboratory of Inflammation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
出版信息
Methods Mol Biol. 2011;689:163-81. doi: 10.1007/978-1-60761-950-5_10.
Abstract
Eicosanoids (prostaglandins, leukotrienes and lipoxins) are a family of signaling lipids derived from arachidonic acid that have important roles in physiological and pathological processes. Over the past years, it has been established that successful eicosanoid production is not merely determined by arachidonic acid and eicosanoid-forming enzymes availability, but requires sequential interactions between specific biosynthetic proteins acting in cascade and may involve very unique spatial interactions. Direct assessment of specific subcellular locales of eicosanoid synthesis has been elusive, as those lipid mediators are newly formed, not stored and often rapidly released upon cell stimulation. In this chapter, we discuss the EicosaCell protocol for intracellular detection of eicosanoid-synthesizing compartments by means of a strategy to covalently cross-link and immobilize the lipid mediators at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid.
摘要
类二十烷酸（前列腺素、白三烯和脂氧素）是一类由花生四烯酸衍生而来的信号脂质，在生理和病理过程中发挥着重要作用。在过去的几年里，已经明确成功合成类二十烷酸不仅取决于花生四烯酸和类二十烷酸形成酶的可用性，还需要参与级联反应的特定生物合成蛋白之间的顺序相互作用，并且可能涉及非常独特的空间相互作用。由于这些脂质介质是新形成的，不被储存，并且在细胞受到刺激时常常迅速释放，因此对类二十烷酸合成的特定亚细胞位点进行直接评估一直难以实现。在本章中，我们将讨论EicosaCell方案，该方案通过一种策略来共价交联并固定脂质介质在其合成位点，然后基于免疫荧光对靶向类二十烷酸进行定位，从而在细胞内检测类二十烷酸合成区室。
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