| A Simple Microplate Assay for Reactive Oxygen Species Generation and Rapid Cellular Protein Normalization
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Author:
Date:
2021-01-05
[Abstract] 2',7'-dichlorofluorescein (DCF) and derivatives are commonly used as fluorescent indicators of a broad spectrum of reactive oxygen species (ROS) generation in cell-based assays. However, there are numerous challenges inherent to the utilization of DCF probes for intracellular microscopic analysis, including bleaching and probe efflux. Plate spectroscopy is comparatively simple and scalable compared to microscopy or flow cytometry-based acquisition, however is often subject to artefacts, including those introduced by thermal gradients and normalization methods. In this protocol we demonstrate a simple and sensitive plate spectrometry-based protocol utilizing the probes H2DCFDA and sulforhodamine B. The truncated sulforhodamine B assay (SRB) for cellular protein allows for a ...
[摘要] [摘要] 2'，7'-二氯荧光素（DCF）及其衍生物在基于细胞的测定中通常用作产生广泛活性氧（ROS）的荧光指示剂。然而，利用DCF探针进行细胞内显微分析存在许多固有的挑战，包括光稳定性和探针外排。与基于显微镜或基于流式细胞仪的采集相比，板式光谱仪相对简单且可扩展，但是通常会受到伪影的影响，包括通过热梯度和归一化方法引入的伪影。在此方案中，我们展示了一种使用探针H 2 DCFDA和磺基若丹明B的简单而灵敏的基于板光谱的方案。细胞蛋白的快速磺基若丹明B检测（SRB）可以对总细胞群体进行稳定的终点测量，同时还可以保留形态，可以与任何其他检测结合或并行进行以对细胞质量进行归一化，并通过显微镜评分进行补充细胞数和核数。氧化应激和归一化方法可能会增强研究细胞分化，应激或毒性的研究领域。。
图形概要：
ROS生成和细胞蛋白定量的图形概述
关键词：二氯，丹明，DCF，SRB，氧化应激，活性氧，测定
[背景]微孔板检测提供比基于显微镜的分析一个显著的优势对于吞吐量的分析设备，优化和分析时间成本。荧光探针（如2'，7'-二氯二氢荧光素二乙酸盐（H 2 ...
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| Rapid Generation of Human Neuronal Cell Models Enabling Inducible Expression of Proteins-of-interest for Functional Studies
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Author:
Date:
2020-05-05
[Abstract] CRISPR-Cas9 technology has transformed the ability to edit genomic sequences and control gene expression with unprecedented ease and scale. However, precise genomic insertions of coding sequences using this technology remain time-consuming and inefficient because they require introducing adjacent single-strand cuts through Cas9 nickase action and invoking the host-encoded homology-directed repair program through the concomitant introduction of large repair templates. Here, we present a system for the rapid study of any protein-of-interest in two neuronal cell models following its inducible expression from the human AAVS1 safe harbor locus. With lox-flanked foundation cassettes in the AAVS1 site and a tailor-made plasmid for accepting coding sequences-of-interest in ...
[摘要] [摘要] CRISPR-Cas9技术以前所未有的简便性和规模改变了编辑基因组序列和控制基因表达的能力。但是，由于需要引入相邻的单链，因此使用该技术进行精确的基因组编码插入仍然很耗时且效率低下。通过减少Cas9切口酶的作用并通过同时引入大型修复模板来调用宿主编码的同源性指导的修复程序。在此，我们提出了一种系统，用于在其诱导后的两个神经元细胞模型中快速研究任何目的蛋白该系统可从人类AAVS1 安全港基因座表达，在AAVS1 位点具有lox侧翼的基础盒和定制的质粒以接受感兴趣的编码序列，该系统使研究人员能够为诱导型产生自己的神经元细胞模型任何编码表达序列不到一个月的时间。由于可用性Preinserted 增强型绿色的Fluo 可以与目标蛋白质融合的最新蛋白质（EGFP）编码序列，该系统可帮助功能研究通过活细胞显微镜以及利用非常有效的可用性进行的相互作用组分析来跟踪目标蛋白质EGFP捕获矩阵。
[背景] ...
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