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Dimethyl sulfoxide

二甲基亚砜

Company: Sigma-Aldrich
Catalog#: D2650
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An Image-based Assay for High-throughput Analysis of Cell Proliferation and Cell Death of Adherent Cells
Author:
Date:
2018-05-05
[Abstract]  In this protocol, we describe a method to monitor cell proliferation and death by live-cell imaging of propidium iodide (PI)-stained adherent mammalian cells. PI is widely used to assess cell death. However, it is usually used in end-point assays. Recently, we implemented the use of PI for real-time cell death assessment by automated imaging. Cells are seeded in a 96-well format, and after attachment, the treatments are added directly to the wells together with PI. Thereafter, cells are subjected to automated time-lapse imaging and quantification by computer software. Combined analyses of phase-contrast and fluorescence images allow assessment of treatment effects on cell proliferation as well as the extent and kinetics of cell death. [摘要]  在该协议中,我们描述了通过碘化丙啶(PI)染色的贴壁哺乳动物细胞的活细胞成像来监测细胞增殖和死亡的方法。 PI广泛用于评估细胞死亡。 但是,它通常用于终点检测。 最近,我们通过自动成像实现了PI的实时细胞死亡评估。 将细胞以96孔形式接种,并且在附着后,将处理与PI一起直接加入到孔中。 之后,通过计算机软件对细胞进行自动延时成像和定量。 相衬和荧光图像的组合分析允许评估对细胞增殖的处理效果以及细胞死亡的程度和动力学。

【背景】多种基于细胞的测定可用于确定细胞死亡,但其中大多数测定包括MTT(溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓)测定,晶蓝染色,和各种基于流式细胞术的方法,都有作为终点分析的限制。最近,我们使用自动荧光成像系统(Essen Bioscience的IncuCyte ZOOM)(Sehgal等人,2017)将碘化丙啶(PI)用于细胞死亡的活细胞评估。通过对相差图像进行组合分析,可以同时检测细胞抑制和细胞毒性效应。这种方法证明是可靠和可重复的,以及非常简单和便宜。在我们最近的出版物中,我们将它用于三种不同的癌细胞系(LNCaP,PC3和MCF7),以有效地确定和比较ER Ca 2 +泵抑制剂毒胡萝卜素各种药物类似物(Tg )(Sehgal et ...

The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines
Author:
Date:
2018-05-05
[Abstract]  Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly dynamic and complex process. Changes in the expression and/or localisation of autophagy-related proteins are frequently assessed by Western blot and various microscopy techniques. Such analyses may be indicative of alterations in autophagy-related processes and informative about the specific marker being investigated. However, since these proteins are part of the autophagic machinery, and not autophagic cargo, they cannot be used to draw ... [摘要]  自噬是维持真核生物细胞稳态的关键因素,包括癌症和神经退行性疾病在内的许多疾病都与自噬的改变有关。研究自噬的兴趣在过去的二十年里急剧增长,并且还有用于研究这种高度动态和复杂过程的方法库。通常通过Western印迹和各种显微镜技术来评估自噬相关蛋白的表达和/或定位中的变化。这样的分析可能表明自噬相关过程的改变,并且关于正在研究的特定标记物的信息。然而,由于这些蛋白质是自噬机制的一部分,而不是自噬性货物,所以它们不能用于得出关于自噬载货流量的结论。在这里,我们提供了一个协议,通过使用长寿命的蛋白质降解测定来定量评估体积自噬流量。我们的程序追踪14 C缬氨酸标记的蛋白质的降解是简单和快速的,允许并行处理相对大量的样品,并且原则上可以与任何贴壁细胞一起使用线。最重要的是,它可以通过自噬途径定量测量内源货物流量。因此,它是研究自噬活动的黄金标准之一。

【背景】脉冲追踪标记方法已用于研究蛋白质周转数十年。在此处描述的长寿命蛋白质降解(LLPD)测定中,培养细胞的蛋白质组用14 ...

Measurement of Intracellular ROS in Caenorhabditis elegans Using 2’,7’-Dichlorodihydrofluorescein Diacetate
Author:
Date:
2018-03-20
[Abstract]   Reactive oxygen species (ROS) are generated during normal metabolic processes under aerobic conditions. Since ROS production initiates harmful radical chain reactions on cellular macromolecules, including lipid peroxidation, DNA mutation, and protein denaturation, it has been implicated in a wide spectrum of diseases such as cancer, cardiovascular disease, ischemia-reperfusion and aging. Over the past several decades, antioxidants have received explosive attention regarding their protective potential against these deleterious reactions. Accordingly, many analytical methodologies have been developed for the evaluation of the antioxidant capacity of compounds or complex biological samples. Herein, we introduce a simple and convenient method to detect in vivo intracellular ROS ... [摘要]  在有氧条件下的正常代谢过程中产生活性氧物质(ROS)。 由于ROS产生会引起细胞大分子的有害自由基连锁反应,包括脂质过氧化反应,DNA突变和蛋白质变性,因此它已涉及多种疾病,如癌症,心血管疾病,缺血再灌注和衰老。 在过去的几十年中,抗氧化剂已经受到爆炸性的关注,因为它们对这些有害反应具有保护作用。 因此,已经开发了许多分析方法用于评估化合物或复杂生物样品的抗氧化能力。 在此,我们介绍了使用2',7'-二氯荧光素二乙酸盐(H 2),在线虫体内用光度计检测体内细胞内ROS水平的简单方便的方法。 DCFDA),一种细胞渗透性示踪剂。

【背景】已经广泛地使用荧光探针2',7'-二氯荧光素二乙酸酯(H 2 DCFDA)原位检测活体内细胞内活性氧簇(ROS)水平由在氧化应激和相关疾病领域工作的研究人员提供。非极性和非离子型探针H 2 DCFDA可以容易地穿透细胞膜并且被酯酶酶促脱乙酰化。该生物化学反应将H 2 DCFDA转化成非荧光化合物H 2 DCF,然后将其迅速氧化成高度荧光的2',7'-二氯荧光素(DCF) ROS的存在(图1A)。因此,来自H 2 ...

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