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Improved multiparametric scrape loading-dye transfer assay for a simultaneous high-throughput analysis of gap junctional intercellular communication, cell density and viability.
Scientific Reports ( IF 3.8 ) Pub Date : 2020-01-20 , DOI: 10.1038/s41598-020-57536-3 Aneta Dydowiczová 1 , Ondřej Brózman 1 , Pavel Babica 1 , Iva Sovadinová 1
Scientific Reports ( IF 3.8 ) Pub Date : 2020-01-20 , DOI: 10.1038/s41598-020-57536-3 Aneta Dydowiczová 1 , Ondřej Brózman 1 , Pavel Babica 1 , Iva Sovadinová 1
Affiliation
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Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJIC represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. This multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJIC, cell density and viability. Our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJIC and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds.
中文翻译：
改进的多参数刮擦加载染料转移测定，可同时对间隙连接细胞间通讯、细胞密度和活力进行高通量分析。
间隙连接细胞间通讯 (GJIC) 是维持组织稳态所需的重要细胞过程。GJIC 的体外评估代表了有价值的表型终点，可以有效地用作现代毒性测试、药物筛选或生物医学体外研究的一个组成部分。然而，目前可用的高通量定量 GJIC 方法通常需要专门的设备、专有软件和/或基因工程细胞模型。为了克服这些限制，我们在此介绍了一种对传统的、基于荧光显微镜的刮擦加载染料转移 (SL-DT) 测定的创新适应，该测定已被优化以同时评估 GJIC、细胞密度和活力。这种多参数方法被证明适用于各种多孔微孔板格式，这有助于自动化图像采集。分析工作流程得到基于开源软件工具的进一步协助，用于批量图像处理、分析和评估 GJIC、细胞密度和活力。我们的结果表明，这种方法为 GJIC 和其他相关表型细胞事件的体外高通量评估提供了一种简单、快速、通用且具有成本效益的方法，可以将其纳入药理学和毒理学相关化合物的体外筛选和评估中。
更新日期：2020-01-21
中文翻译：
改进的多参数刮擦加载染料转移测定，可同时对间隙连接细胞间通讯、细胞密度和活力进行高通量分析。
间隙连接细胞间通讯 (GJIC) 是维持组织稳态所需的重要细胞过程。GJIC 的体外评估代表了有价值的表型终点，可以有效地用作现代毒性测试、药物筛选或生物医学体外研究的一个组成部分。然而，目前可用的高通量定量 GJIC 方法通常需要专门的设备、专有软件和/或基因工程细胞模型。为了克服这些限制，我们在此介绍了一种对传统的、基于荧光显微镜的刮擦加载染料转移 (SL-DT) 测定的创新适应，该测定已被优化以同时评估 GJIC、细胞密度和活力。这种多参数方法被证明适用于各种多孔微孔板格式，这有助于自动化图像采集。分析工作流程得到基于开源软件工具的进一步协助，用于批量图像处理、分析和评估 GJIC、细胞密度和活力。我们的结果表明，这种方法为 GJIC 和其他相关表型细胞事件的体外高通量评估提供了一种简单、快速、通用且具有成本效益的方法，可以将其纳入药理学和毒理学相关化合物的体外筛选和评估中。
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