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Fetal bovine serum (FBS)

胎牛血清

Company: Thermo Fisher Scientific
Catalog#: 10270106
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Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network
Author:
Date:
2020-03-05
[Abstract]  Protein sorting at the trans Golgi network (TGN) plays important roles in targeting newly synthesized proteins to their specific destinations. The aim of this proposal is to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN, utilizing rat liver cytosol, semi-intact mammalian cells and nucleotides. The protocol describes how to perform the vesicle formation assay, how to isolate vesicles and how to detect cargo proteins in vesicles. This reconstitution assay can be used to quantitatively measure the efficiency of the packaging of a specific cargo protein into transport vesicles at the TGN under specific experimental conditions. [摘要]  [摘要] 反式高尔基体网络(TGN)上的蛋白质分选在将新合成的蛋白质靶向其特定目的地方面起着重要作用。该提议的目的是利用大鼠肝细胞溶质,半完整的哺乳动物细胞和核苷酸,在TGN处将非高尔基驻留的货物蛋白重新包装成囊泡。该协议描述了如何进行囊泡形成测定,如何分离囊泡以及如何检测囊泡中的货物蛋白。该重构测定法可用于定量测量在特定实验条件下将特定货物蛋白包装到TGN的运输小泡中的效率。

[背景] 的反式高尔基体网络(TGN)是在分泌运送路径的必要的交通枢纽。为了确保水泡运输的保真度,真核细胞利用各种蛋白质分选设备将特定的货物蛋白质准确地包装到TGN的运输小泡中,然后运至特定的目的地(Guo 等人,2014)。为了加深我们对TGN分选过程特异性的理解,重要的是开发一种能够忠实地重构TGN囊泡形成和货物分选过程的分析方法。该测定法可用于直接和定量地测量特定因子在调节特定货物蛋白包装到运输小泡中的作用。从内质网(ER)将货物蛋白包装到COPII囊泡中的无细胞重构已得到很好的建立(Kim 等,2005; Kim 等,2007; Merte 等,2010; Yuan 等。,2018;Niu 等,2019;)。已经开发出一种体外测定法,其在TGN处重构特定货物蛋白TGN46在运输小泡中的释放(Ponnambalam 等,1996;Wakana ...

Isolation and Culture of Mouse Lung ILC2s
Author:
Date:
2018-10-05
[Abstract]  Group 2 Innate Lymphoid Cells (ILC2) play an important role in immune responses at barrier surfaces, notably in the lung during airway allergic inflammation or asthma. Several studies have described methods to isolate ILC2s from wild-type naive mice, most of them using cell sorting to obtain a pure population. Here, we describe in detail, a simple, efficient method for isolation and culture of lung mouse ILC2s. Lungs from Rag2-/- mice pretreated with IL-33 are collected and processed into single cell suspensions. Lymphoid cells are then recovered by density gradient separation. Lin-CD45+ cells are selected by depletion of lineage positive cells followed by positive selection of CD45+ cells. Culture of the isolated cells for several days ... [摘要]  第2组先天性淋巴细胞(ILC2)在屏障表面,特别是在气道过敏性炎症或哮喘期间的肺中的免疫应答中起重要作用。一些研究已经描述了从野生型幼稚小鼠中分离ILC2的方法,其中大多数使用细胞分选来获得纯种群。在这里,我们详细描述了一种简单有效的肺小鼠ILC2分离和培养方法。收集用IL-33预处理的 Rag2 - / - 小鼠的肺并加工成单细胞悬浮液。然后通过密度梯度分离回收淋巴样细胞。通过耗尽谱系阳性细胞然后阳性选择CD45 + 细胞来选择Lin - CD45 + 细胞。将分离的细胞培养数天导致高度纯化的ILC2群体表达典型的细胞表面标志物(CD90.2,Sca1,CD25,CD127和IL-33R)。这些细胞可在培养物中扩增长达10天,并用于多种离体测定或体内过继转移实验。
【背景】第2组先天性淋巴细胞(ILC2)是组织驻留细胞,其在抗寄生虫先天免疫以及过敏性炎症的发展中起关键作用。它们通过产生大量的2型细胞因子IL-5和IL-13对上皮细胞衍生的细胞因子如白细胞介素-33(IL-33)起反应,后者又诱导嗜酸性粒细胞增多和粘液产生(Cayrol和Girard,2018)。为了更好地表征这些细胞的功能和调节,许多组通过荧光激活细胞分选(FACS)从野生型小鼠(WT)的肺中分选ILC2。由于稳定状态下肺中存在的ILC2数量较少,因此该方法导致纯化细胞的产量较低(每只小鼠1×10 ...

Zebrafish Embryo Xenograft and Metastasis Assay
Author:
Date:
2018-09-20
[Abstract]  Xenograft models, and in particular the mouse xenograft model, where human cancer cells are transplanted into immunocompromised mice, have been used extensively in cancer studies. Although these models have contributed enormously to our understanding of cancer biology, the zebrafish xenograft model offers several advantages over the mouse model. Zebrafish embryos can be easily cultured in large quantities, are small and easy to handle, making it possible to use a high number of embryos for each experimental condition. Young embryos lack an efficient immune system. Therefore the injected cancer cells are not rejected, and the formation of primary tumors and micrometastases is rapid. Transparency of the embryos enables imaging of primary tumors and metastases in an intact and living embryo. ... [摘要]  异种移植模型,特别是小鼠异种移植模型,其中人癌细胞被移植到免疫受损小鼠中,已广泛用于癌症研究。尽管这些模型对我们对癌症生物学的理解做出了巨大贡献,但斑马鱼异种移植模型与鼠标模型相比具有多种优势。斑马鱼胚胎可以很容易地大量培养,体积小且易于处理,因此可以在每种实验条件下使用大量胚胎。年轻的胚胎缺乏有效的免疫系统。因此,注射的癌细胞不被排斥,并且原发性肿瘤和微转移的形成是快速的。胚胎的透明度使得能够在完整和活的胚胎中对原发性肿瘤和转移瘤进行成像。在这里,我们描述了一种方法,其中表达GFP的肿瘤细胞被注射到斑马鱼胚胎的心包空间。在注射后4天,对胚胎成像并分析原发性肿瘤和远端微转移的形成。

【背景】 斑马鱼( Danio rerio )是一种小型淡水鱼,不仅在发育生物学中,而且在生物医学中越来越受欢迎。快速发展的领域之一是使用斑马鱼作为癌症生物学研究的模型。斑马鱼高效可靠地交配,可以产生大量小,透明,外部发育的后代,可以在例如,多孔板中轻松培养(White et al。 >,2013)。有趣的是,人类肿瘤细胞可以植入斑马鱼胚胎中,许多异种移植物能够通过在胚胎中生长,侵入和转移来忠实地重演它们的恶性行为(Lee et al。,2005; Nicoli et al。,2007; Chapman et ...

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