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Fetal Bovine Serum, certified, US origin

胎牛血清

Company: Thermo Fisher Scientific
Catalog#: 16000044
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Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
Author:
Date:
2018-01-05
[Abstract]  Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary cortical neurons by Western blotting. Our protocol is based on reducing the amounts of BSA in the media while maintaining cell viability and secretory potential, and concentrating the media prior to analysis by means of ultrafiltration. [摘要]  由细胞分泌的蛋白质的定量分析由于它们的丰度低和在细胞培养基中干扰大量牛血清白蛋白(BSA)的存在而提出挑战。 我们建立检测突变亨廷顿蛋白(mHtt)检测分泌Neuro2A细胞系稳定表达mHtt和大鼠原代皮层神经元蛋白质印迹。 我们的方案是基于降低培养基中BSA的量,同时保持细胞活力和分泌潜能,并在通过超滤分析之前浓缩培养基。


【背景】许多蛋白质通过各种分泌途径从细胞分泌到细胞外环境中。这些途径包括在ER-高尔基体 - 质膜途径之后的常规分泌途径(Lee等,2004)和多个非常规途径,例如溶酶体胞吐作用,穿过质膜的易位和外泌体以及胞外体释放(Zhang和Schekman,2013)。为了研究这些途径,经常需要分析培养细胞分泌的蛋白质进入培养基。蛋白质可以游离形式分泌,也可以与胞膜结构如胞外体和外泌体结合(Zhang and ...

High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
Author:
Date:
2018-01-05
[Abstract]  Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal 3H-serotonin secretion assay. The NPY-Venus secretion assay was successfully used for a high throughput siRNA screen, and the serotonin secretion assay was used to validate hits identified from the screen (Sorensen, 2017; Zhang et al., 2017). [摘要]  在这里我们描述了两种测定方法来测量神经内分泌细胞中的致密核心囊泡(DCV)胞吐作用介导的货物分泌。 为了在受调控的DCV胞吐作用中对新基因进行siRNA筛选,我们开发了使用DCV货物,NPY-金星以及正交3H-血清素分泌测定的基于读板器的分泌测定法。 NPY-金星分泌测定成功地用于高通量siRNA筛选,并且使用血清素分泌测定来验证从筛选中鉴定的命中(Sorensen,2017; Zhang等人,2017)。


【背景】致密核心囊泡(DCV)胞吐作用介导内分泌和神经内分泌细胞分泌蛋白质,肽和小分子。当蛋白质和肽激素被内质网上的核糖体合成时,蛋白质和肽激素进入分泌途径,并在跨高尔基网络(TGN)(Borgonovo等人,2006; Bowman等, >等人,2009)。小分子物质,如5-羟色胺,直接被胞质单胺转运蛋白(VMATs)从细胞质直接吸收到DCV的内腔(Sudhof,2004)。在非刺激条件下,货物分子被储存在靠近质膜的DCV内。当细胞接受增加细胞质Ca ...

Isolation, BODIPY Labeling and Uptake of Exosomes in Hepatic Stellate Cells
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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