| Triacylglycerol Measurement in HeLa Cells
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Author:
Date:
2020-12-20
[Abstract] Lipid droplets store triacylglycerols (triglycerides) and sterol esters to regulate lipid and energy homeostasis. Triacylglycerol measurement is often performed during the investigation of lipid droplet formation and growth. This protocol describes a reliable method using a fluorometric lipid quantification kit to measure triacylglycerols extracted from HeLa cells, which were treated with oleic acid to trigger the formation of lipid droplets. The lipid quantification kit employs a lipid-binding molecule that emits bright fluorescence only when bound to extracted triacylglycerols, whose content can be quantified by a simple fluorescence readout.
[摘要] [摘要]脂滴商店吨riacylglycerols （甘油三酯）和甾醇酯调节脂质和能量稳态。通常在研究脂质滴的形成和生长过程中进行三酰基甘油的测量。该协议描述了一种使用荧光脂质定量试剂盒测量从HeLa细胞提取的三酰基甘油的可靠方法，该甘油经油酸处理后触发了脂质滴的形成。脂质定量试剂盒采用的脂质结合分子仅在与提取的三酰基甘油结合时才发出明亮的荧光，其含量可以通过简单的荧光读数来定量。
[背景]脂滴（LD）的是具有特征功能独特的细胞器。与常见的细胞器不同，每个LD由磷脂的单层定义，该磷脂包裹着中性脂质（如三酰甘油（TAG），也称为甘油三酸酯）和甾醇酯的核心。作为存储细胞器，LDs在脂质和能量的动态平衡以及其他细胞事件中起主要作用。通过以TAG的形式存储过量的脂肪酸，LDs可以防止脂毒性并保护细胞免受氧化应激。重要的是，LDs几乎与所有其他细胞器接触并调节关键的细胞过程，包括膜运输，蛋白质更新和病毒感染（Olzmann和Carvalho，2018; Gao等，2019）。此外，LD表面装饰有各种蛋白质，这些蛋白质参与TAG的合成和水解以及LD与其他细胞器之间的脂质运输（Kory等，2016; Kumar等，2018; Du等。 。，2020）。
真核细胞具有完善的途径来合成LDs中存储的TAG ...
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| Human, Bacterial and Fungal Amplicon Collection and Processing for Sequencing
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Author:
Date:
2015-05-20
[Abstract] Sequencing taxonomic marker genes is a powerful tool to interrogate the composition of microbial communities. For example, bacterial and fungal community composition can be evaluated in parallel using the 16S ribosomal RNA gene for bacteria or the internal transcribed spacer region in fungi. These are conserved regions that are universal to a taxonomic clade, yet have undergone some degree of evolution such that different lineages can be differentiated. Conserved regions are used for design of universal priming sites that allow amplification of the marker gene out of a mixed microbial community. Here, we describe our standard operating procedure to collect and sequence 16S rRNA and ITS1 amplicons from human skin. We use the 16S rRNA V1-V3 region for skin samples, as it has greater power ...
[摘要] 测序分类标记基因是一个强有力的工具，以询问微生物群落的组成。 例如，可以使用细菌的16S核糖体RNA基因或真菌中的内部转录间隔区平行评估细菌和真菌群落组成。 这些是对分类学分支是通用的保守区，但已经经历了某种程度的进化，使得不同谱系可以分化。 保守区用于设计允许从混合微生物群落扩增标记基因的通用引发位点。 在这里，我们描述我们的标准操作程序收集和序列16S rRNA和ITS1扩增子从人类皮肤。 我们使用16S rRNA V1-V3区域的皮肤样本，因为它有更大的权力分类皮肤中的常见葡萄球菌。 该方案适用于扩增子的454焦磷酸测序。
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