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HEK293 cells

HEK-293

Company: ATCC
Catalog#: CRL-1573
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Quantification of the Surface Expression of G Protein-coupled Receptors Using Intact Live-cell Radioligand Binding Assays
Author:
Date:
2020-09-20
[Abstract]  G protein-coupled receptors (GPCRs) are the most structurally diverse family of signaling proteins and regulate a variety of cell function. For most GPCRs, the cell surface is their functional destination where they are able to respond a wide range of extracellular stimuli, leading to the activation of intracellular signal transduction cascades. Thus, the quantity of receptor expression at the cell surface is a crucial factor regulating the functionality of the receptors. Over the past decades, many methods have been developed to measure the cell surface expression of GPCRs. Here, we describe an intact live-cell radioligand binding assay to quantify the surface expression of GPCRs at the endogenous levels or after overexpression. In this assay, cell cultures will be incubated with ... [摘要]  [摘要] G蛋白偶联受体(GPCR)是信号蛋白中结构最多样化的家族,可调节多种细胞功能。对于大多数GPCR,细胞表面是它们的功能目的地,能够响应广泛的细胞外刺激,从而激活细胞内信号转导级联反应。因此,受体在细胞表面的表达量是调节受体功能的关键因素。在过去的几十年中,已开发出许多方法来测量GPCR的细胞表面表达。在这里,我们描述了完整的活细胞放射性配体结合测定法,以量化内源水平或过表达后GPCR的表面表达。在该测定中,将细胞培养物与特定的细胞不可渗透的放射性配体温育,所述放射性不可配体选择性地和化学计量地结合至各个GPCR,并且通过受体结合的配体的放射性来定量细胞表面的受体数量。 此方法对于测量完整活细胞表面的功能性GPCR具有高度特异性,对于内源性,低丰度的GPCR特别有用。

[背景 ] G蛋白偶联受体(GPCR)构成细胞表面受体的最大超家族,并在生理和病理条件下调节多种细胞功能(Hauser 等人,2017; Hilger 等人,2018; Weinberg和Puthenveedu,2019) ...

Assessing Gαq/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay
Author:
Date:
2020-08-20
[Abstract]  Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gαq/15-coupled receptors rely on the use of fluorescent probes for Ca++ sensing (such as Fura-2 and Fluo-4) or on the incorporation of [3H]-inositol into inositol 1,4,5- triphosphate (IP3). However, these methods are not suitable for screening large libraries of compounds or for screening several mutants of the same receptor. In contrast, the IP-One assay by Cisbio is a TR-FRET assay suitable for large compound library screening when using stable cell lines that express ... [摘要]  [摘要 ] 基于细胞的功能测定法是化合物筛选和药物先导物优化的重要组成部分,并且它们可以也参与配体结合和信令残留量的测定起到至关重要的作用为一个特定的G蛋白偶联受体。用于Gα常规方法的q / 15 -偶联受体依赖于使用用于钙荧光探针++ 感测(如的Fura-2和的Fluo-4)或上掺入[的3 H] - 肌醇成肌醇1,4- ,5-三磷酸(IP3)。然而,这些方法不适合用于筛选大文库的化合物或用于筛选相同的受体的几个突变体。相反,IP-一个测定由Cisbio公司是TR-FRET测定适合大型化合物文库使用的稳定细胞株的筛选时,表达一个特定7TMR 。但是,当使用瞬时转染的7TMR突变体时,此检测方法并不理想,因为它需要两步操作进行细胞培养。因此,我们已经优化了IP-One的测定使用协议的在384孔反向转染方法的板。这为先前用于筛选Gαq / 15 偶联7TMR 的几种突变体的两步法提供了一种省时和省资源的替代方案。

[背景 ] 七跨膜受体(7TMR),也被称为G蛋白偶联受体,是超家族参与信号转导的最重要的跨膜蛋白。它们是临床批准药物的30%至50%的目标(Overington 等,2006)。Gαq / 15 偶联的7TMR激活磷脂酶Cβ(PLCβ),并产生D-肌醇1,4,5-三磷酸(IP3)和二酰基甘油(DAG)。IP3触发细胞内Ca ++ ...

Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells
Author:
Date:
2018-09-05
[Abstract]  MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with low resolution for the complexity of the miRNA-induced regulation. Here, we provide a protocol using a dual-fluorescence reporter and flow cytometry reaching single cell resolution; the protocol contains a simplified workflow that includes: vector generation, data acquisition, processing, and analysis using the R environment. Our protocol enables high-resolution measurements of miRNA induced post-transcriptional gene regulation and combined with ... [摘要]  MicroRNA诱导的基因调控是基础和转化研究中不断增长的领域。 直接在细胞中检查该调节对于验证源自RNA测序技术的高通量数据是必要的。 对于这一研究,一些研究采用基于荧光素酶的报告基因,通常测量全细胞群,其具有低分辨率的miRNA诱导调节的复杂性。 在这里,我们提供使用双荧光报告基因和流式细胞仪达到单细胞分辨率的方案; 该协议包含一个简化的工作流程,包括:使用R环境进行矢量生成,数据采集,处理和分析。 我们的协议可实现miRNA诱导的转录后基因调控的高分辨率测量,并结合系统生物学,可用于估计miRNA的熟练程度。

【背景】MicroRNAs(miRNA)是高度保守的小型非蛋白质编码RNA(21-22nt),可调节转录后基因表达并调节基因生物学过程,如发育和细胞稳态(Lagos-Quintana et al。,2001; Fabian et al。,2010; Bartel,2018),包括miRNA表达与肿瘤进展和侵袭性相关的几种病理(Lu et al。, 2005; Di Leva和Croce,2013; Krishnan et al。,2015; Bertoli et ...

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