Valeria Di Gioia, Jan Zamporlini, Rebecca Vadalà, Elena Parmigiani, Beatrice Bodega, Federica Marasca
{"title":"分析各种细胞类型核体动态变化的多功能管道","authors":"Valeria Di Gioia, Jan Zamporlini, Rebecca Vadalà, Elena Parmigiani, Beatrice Bodega, Federica Marasca","doi":"10.3791/66874","DOIUrl":null,"url":null,"abstract":"<p><p>Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience. In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution. Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different contexts.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2000,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types.\",\"authors\":\"Valeria Di Gioia, Jan Zamporlini, Rebecca Vadalà, Elena Parmigiani, Beatrice Bodega, Federica Marasca\",\"doi\":\"10.3791/66874\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience. In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution. Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different contexts.</p>\",\"PeriodicalId\":48787,\"journal\":{\"name\":\"Jove-Journal of Visualized Experiments\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.2000,\"publicationDate\":\"2024-06-28\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Jove-Journal of Visualized Experiments\",\"FirstCategoryId\":\"103\",\"ListUrlMain\":\"https://doi.org/10.3791/66874\",\"RegionNum\":4,\"RegionCategory\":\"综合性期刊\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"MULTIDISCIPLINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Jove-Journal of Visualized Experiments","FirstCategoryId":"103","ListUrlMain":"https://doi.org/10.3791/66874","RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
引用次数: 0
摘要
各种核过程(如转录控制)都发生在被称为病灶的离散结构中,通过免疫荧光技术可以分辨出这些病灶。通过显微镜研究这些病灶在不同细胞条件下的动态变化,可以深入了解细胞特性和功能的分子机制。然而,在不同类型的细胞中进行免疫荧光检测,并评估这些病灶的组装、扩散和分布变化,却面临着诸多挑战。这些挑战包括样品制备的复杂性、成像数据分析参数的确定以及大量数据的管理。此外,现有的成像工作流程通常是为熟练用户量身定制的,因此限制了更多人的使用。在本研究中,我们介绍了一种优化的免疫荧光方案,专门用于研究不同人类原代 T 细胞类型中的核蛋白,该方案可针对任何感兴趣的蛋白质和细胞类型进行定制。此外,我们还介绍了一种无偏量化蛋白质染色的方法,无论它们是形成明显的病灶还是呈现弥散的核分布。我们提出的方法提供了从细胞染色到分析的全面指导,利用 Jython 开发的半自动化管道,可在 Fiji 中执行。此外,我们还提供了一个用户友好型 Python 脚本,以简化数据管理,该脚本可在 Google Colab 笔记本上公开访问。我们的方法在对不同背景下具有不同核组织模式的蛋白质进行高信息量的免疫荧光分析方面证明了其有效性。
A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types.
Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience. In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution. Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different contexts.
期刊介绍:
JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.