|
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 ...
|
|
Author:
Date:
2016-11-05
[Abstract] The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be extracted using basic conditions; thus, the amount of bound dye can be used as a proxy for cell mass, which can then be extrapolated to measure cell proliferation.
The protocol can be divided into four main steps: preparation of treatment, incubation of cells with treatment of choice, cell fixation and SRB staining, and absorbance measurement. This assay is limited to manual or semiautomatic screening, and can be used in an efficient ...
[摘要] Limonium 已知有性和无融合生殖（通过种子的无性繁殖）繁殖模式。在这里，我们提出解剖协议为胚珠的发芽。使用微分干涉对比（DIC）显微镜。该协议允许更好地处理胚珠，并提供优于早期技术，特别是在较大胚珠的某些优势。这种方法还能够观察到完整胚珠中的减数分裂和胚囊发育，以及容易检测到区别性和无序性发育的事件。
[背景] 发生在胚珠发育期间，有必要细胞学检查胚珠。这项研究可以涉及显微镜观察石蜡或树脂包埋，切片材料或清除的器官。在D'Amato（1940; 1949）的先驱作品中公开了对性和无融合生殖物种中胚珠和胚囊发育的第一次细胞学研究。在这些作品中，花使用Karpechenko的方法固定，包埋在石蜡中，切片并用Heidenhain的铁苏木精染色，其染色质和染色体在细胞核中染色。使用这些方法的花芽切片可导致由于单个细胞的部分破坏结构完整性而具有差质量的制备物。更容易的选择是清除福尔马林：乙酸：乙醇固定的器官并用纯的Mayer's hemalum染色（Wallis，1957; Stelly等人，1984）。这种技术需要少得多的时间和劳动，特别是对于通常在卵巢中形成小胚珠的物种，这是 Limonium ...
|