{"title":"用流式细胞仪定量测量异源细胞样本中的质膜蛋白内化和再循环。","authors":"Hui Jing Lim, Hamish E G McWilliam","doi":"10.21769/BioProtoc.4986","DOIUrl":null,"url":null,"abstract":"<p><p>Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry-based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level. Key features • These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry. • They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available. • The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states. • The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells. • These assays have been extensively used on suspension cells but have not been tested on adherent cells.</p>","PeriodicalId":1,"journal":{"name":"Accounts of Chemical Research","volume":null,"pages":null},"PeriodicalIF":16.4000,"publicationDate":"2024-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11082785/pdf/","citationCount":"0","resultStr":"{\"title\":\"Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry.\",\"authors\":\"Hui Jing Lim, Hamish E G McWilliam\",\"doi\":\"10.21769/BioProtoc.4986\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. 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引用次数: 0
摘要
质膜蛋白介导着生理学的重要方面,包括营养获取、细胞-细胞相互作用和监测平衡。这些蛋白质的运输,包括从细胞表面的内化和/或循环回到细胞表面,往往对其功能至关重要。这些过程因蛋白质、细胞类型和状态的不同而各异,目前仍在研究之中。目前测量表面蛋白内化和再循环的方法通常是显微镜或生化分析法;这些方法虽然准确,但通常仅限于分析同质细胞群,而且通量较低。在这里,我们介绍了基于流式细胞术的方法,该方法涉及探针连接抗体,可在复杂样本中对单细胞水平的内化或回收率进行量化。为了测量内化率,我们详细介绍了一种检测方法,即用特异性抗体与荧光寡核苷酸标记的探针结合来标记相关蛋白质。为了测量再循环,我们使用了一种与可裂解生物素基团连接的特异性抗体。这些探针可以区分已被内化或回收的分子和未被内化或回收的分子。这些方法与细胞特异性标记板相结合,可在单细胞水平上定量研究异源细胞混合物中质膜蛋白质的贩运动态。主要特点 - 这些检测方法可分别使用寡核苷酸或可裂解生物素连接的探针,通过流式细胞术灵敏地定量检测内化或回收的表面分子。- 它们可适用于任何通过细胞表面转运的膜蛋白,并可获得特异性纯化抗体。- 细胞表面蛋白的动态可在异源细胞群中同时测量,包括各种细胞活化状态。- 内化检测建立在 Liu 等人开发的方法基础上[1,2],并将其应用扩展到异源人类外周血单核细胞。- 这些检测方法已广泛应用于悬浮细胞,但尚未在粘附细胞上进行过测试。
Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry.
Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry-based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level. Key features • These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry. • They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available. • The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states. • The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells. • These assays have been extensively used on suspension cells but have not been tested on adherent cells.
期刊介绍:
Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance.
Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.