| Imaging of Human Cancer Cells in 3D Collagen Matrices
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Author:
Date:
2021-01-20
[Abstract] Research on cell migration and interactions with the extracellular matrix (ECM) was mostly focused on 2D surfaces in the past. Many recent studies have highlighted differences in migratory behaviour of cells on 2D surfaces compared to complex cell migration modes in 3D environments. When embedded in 3D matrices, cells constantly sense the physicochemical, topological and mechanical properties of the ECM and adjust their behaviour accordingly. Changes in the stiffness of the ECM can have effects on cell morphology, differentiation and behaviour and cells can follow stiffness gradients in a process called durotaxis. Here we introduce a detailed protocol for the assembly of 3D matrices consisting of collagen I/fibronectin and embedding cells for live cell imaging. Further, we will show how ...
[摘要] [摘要]细胞迁移及其与细胞外基质（ECM）相互作用的研究最多过去专注于2D曲面。最近的许多研究都强调了与3D环境中复杂的细胞迁移模式相比，2D表面上的细胞迁移行为的差异。当嵌入3D矩阵中时，细胞会不断感知ECM的物理化学，拓扑和机械特性，并相应地调整其行为。ECM刚度的变化会影响细胞的形态，分化和行为，并且细胞会在称为durotaxis的过程中遵循刚度梯度。在这里，我们介绍了由胶原蛋白I /纤连蛋白和包埋细胞组成的3D矩阵的详细协议，用于活细胞成像。此外，我们将展示如何通过非酶糖基化来增强基质，以及用荧光染料对胶原蛋白进行染色如何使基质和细胞同时成像。该方法可用于在具有不同刚度的3D微环境中对细胞迁移进行成像，定义细胞-基质相互作用以及细胞对变化的ECM的反应，并可视化细胞的基质变形。
[背景]细胞和依赖于动态周围的细胞外基质（ECM）生成的功能性实体的调整两者的，基质和细胞，以预防疾病。多年来，人们一直认为ECM仅为嵌入式细胞提供结构支持。但是，最近的研究突出了ECM的关键功能，而不是其脚手架功能。ECM的修饰已与疾病进展相关，尤其是在癌症的背景下，与转移的发生有关，以及与临床预后和患者生存的相关性。
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| 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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