| Lipid Droplet Isolation from Arabidopsis thaliana Leaves
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Author:
Date:
2020-12-20
[Abstract] Lipid droplets (LDs) are neutral lipid aggregates surrounded by a phospholipid monolayer and specific proteins. In plants, they play a key role as energy source after seed germination, but are also formed in vegetative tissues in response to developmental or environmental conditions, where their functions are poorly understood. To elucidate these, it is essential to isolate LDs with good yields, while retaining their protein components. LD isolation protocols are based on their capacity to float after centrifugation in sucrose gradients. Early strategies using stringent conditions and LD-abundant plant tissues produced pure LDs where core proteins were identified. To identify more weakly bound LD proteins, recent protocols have used low stringency buffers, but carryover contaminants and ...
[摘要] [摘要]脂质滴（LDs）是被磷脂单层和特定蛋白质包围的中性脂质聚集体。在植物中，它们在种子发芽后作为能源起着关键作用，但由于发育或环境条件而对其功能了解甚少，它们也在营养组织中形成。为了阐明这些，必须分离出具有高产率的LD，同时保留其蛋白质成分。LD分离方案基于其在蔗糖梯度中离心后的漂浮能力。早期使用严格条件和LD丰富的植物组织的策略产生了可鉴定核心蛋白的纯LD。为了鉴定更弱结合的LD蛋白，最近的研究otocols使用了低严格性的缓冲液，但是残留污染物和低收率通常是个问题。我们已经开发了一种基于蔗糖梯度的方案，可使用Tween-20和新鲜组织从拟南芥叶中分离LD，以提高产量。在健康和细菌感染的拟南芥叶片中，该方案均允许鉴定LD蛋白，随后通过显微镜分析对其进行确认。
背景]脂质降住所（LD）的通过由磷脂单层，其中LD蛋白插入包围的中性脂质核心组成的细胞器（Tzen等人，1993） 。LD首先被描述为稳定的隔室，用于在特殊组织中存储高能脂质。然而，最近的评论将LDs描述为大多数细胞类型中存在的高度动态的细胞器，其功能超越了单纯的能量存储（Shimada等人，2018; Huang，2018; Shao等人，2019）。
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| Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells
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Author:
Date:
2017-12-05
[Abstract] Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with pleiotropic effects from inducing antiviral state to disease progression. We have previously shown that hepatitis C virus (HCV) infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in culture supernatants. These exosomes are taken up by hepatic stellate cells (HSC) and activate them to induce fibrosis during HCV infection. Here, we describe detailed protocols for exosomes isolation and uptake of BODIPY labeled exosomes ...
[摘要] 外来体已经成为细胞间通讯的重要介质。 它们存在于细胞外环境中，因此可以被邻近或远处的细胞轻易获得。 它们在囊泡内携带mRNA，microRNA和蛋白质，并一度被受体细胞内化; 它们可以通过诱导抗病毒状态向疾病进展调节多种信号传导途径，具有多效性。 我们以前已经显示，携带基因组长度复制子的丙型肝炎病毒（HCV）感染的肝细胞或肝癌细胞在培养上清液中分泌外泌体。 这些外来体被肝星状细胞（HSC）摄取，并在HCV感染期间激活它们以诱导纤维化。 在这里，我们描述了外来体分离和肝星状细胞摄取BODIPY标记的外来体的详细的协议。
【背景】外来体是直径为40-150nm的小的膜结合的细胞外囊泡。已在所有体液中鉴定出它们，包括尿，羊水，血清，唾液，母乳，脑脊液，鼻分泌物和组织培养的细胞系的上清液中。外泌体通过转移微小RNA（mRNA），mRNA和蛋白质来介导细胞与细胞之间的通讯。 其可被邻近或远处的细胞摄取，并随后促进受体细胞中的信号传导（Schorey等人，2015）。外泌体富含蛋白质，包括四跨膜蛋白家族成员（CD9，CD81，CD63），热休克蛋白（Hsp60，Hsp70，Hsp90）和多泡体蛋白（annexins，RabGTPases和内体分选复合物，蛋白质）。最近的研究表明，外来体可以影响免疫应答（Li等人，2013; ...
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| BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry
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Author:
Date:
2016-09-05
[Abstract] Lipid droplets (LDs) are ubiquitous, dynamic organelles and function as a storage depot for neutral lipids, including triglycerides and cholesterol esters (Walther and Farese, 2012). The movement of lipid species into and out of LDs impacts a variety of cellular processes, such as energy homeostasis, lipid-based signaling, and membrane homeostasis (Greenberg et al., 2011). For example, neutral lipid storage is enhanced upon increased synthesis or uptake of lipid species. On the other hand, extracellular signals can enhance the release of lipid species packaged within neutral LDs. Thus, the investigation of topics involving lipid metabolism may require the assessment of cellular neutral lipid content. In this protocol, we describe the use of the fluorescent neutral lipid dye ...
[摘要] 脂滴（LD）是普遍存在的，动态的细胞器，并且作为中性脂质包括甘油三酯和胆固醇酯的储存库（Walther和Farese，2012）。 脂质物质进出LD的运动影响多种细胞过程，例如能量稳态，基于脂质的信号传导和膜内环境稳定（Greenberg等人，2011）。 例如，当增加脂质种类的合成或摄取时，中性脂质储存增强。 另一方面，细胞外信号可以增强包装在中性LD内的脂质物质的释放。 因此，涉及脂质代谢的主题的研究可能需要评估细胞中性脂质含量。 在该方案中，我们描述了荧光中性脂质染料4,4-二氟-1,3,5,7,8-五甲基-4-硼-3a，4a-二氮杂-s-引达省（BODIPY 493/503 ），以通过流式细胞术和通过显微镜观察LD来促进中性脂质含量的定量。
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