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6-well plates

Costar ® 6孔透明TC多孔板

Company: Corning
Catalog#: 3506
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NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
Author:
Date:
2017-10-20
[Abstract]  This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from Sambrook and Russell (2001) in order to downscale the amount of cells needed. To determine the efficiency of fractionation, we recommend using qPCR to compare the subcellular compartments that have been purified with equivalent amount of control whole cell extracts. [摘要]  该技术允许在哺乳动物细胞中利用NP-40洗涤剂进行高效,高度纯化的细胞质和核相关的分室分离。 在分离过程中核膜不受干扰,从而使核部分中的所有核和核周相关成分留下。 该协议已经从Sambrook和Russell(2001)修改,以便缩减所需的细胞数量。 为了确定分馏的效率,我们建议使用qPCR来比较已经用等量的对照全细胞提取物纯化的亚细胞室。
【背景】为了充分获得对细胞过程的理解,需要分离核和细胞质隔室。 有许多协议,甚至一些商业套件可用于帮助分离两个隔间。 然而,大多数需要高离心速度,产量差异甚至验证最终产品中的污染物量的方法也是很高的。 我们的协议在低速下使用小型台式离心机,以获得高纯度的细胞质提取物和核/核周组合相关隔室,以及数据分析,以验证污染物的百分比。 迄今为止,已经测试的细胞系是293T,HeLa和GHOST细胞系。 (Galvis,2014; Galvis等,,2014)。

Generation of a Cellular Reporter for Functional BRD4 Inhibition
Author:
Date:
2017-07-05
[Abstract]  The ubiquitously expressed bromodomain-containing protein 4 (BRD4) is an epigenetic reader, which recruits transcriptional regulatory complexes to acetylated chromatin. Because of its role in enhancing proliferation, BRD4 has become a therapeutic target in oncology, as the inhibition of this protein leads to the reduction of the growth of many tumours. Even though BRD4 is more and more studied, its mechanism of action has not been fully described yet. Therefore, we aimed at generating a cellular reporter system to monitor BRD4 inhibition. Such reporter can be potentially used in high throughput chemical and genetic screenings, in order to uncover new possible BRD4 functional pathways. The deeper understanding of the mechanism of action of BRD4 activity will certainly help in developing ... [摘要]  普遍表达的含溴结构域的蛋白质4(BRD4)是表观遗传学读者,其将转录调节复合物募集到乙酰化染色质。 由于其在增强增殖中的作用,BRD4已经成为肿瘤学的治疗靶点,因为抑制这种蛋白质导致许多肿瘤的生长减少。 即使BRD4越来越多的研究,其行动机制尚未得到充分的描述。 因此,我们旨在产生细胞报告系统来监测BRD4抑制。 这种记录可以潜在地用于高通量化学和遗传筛选,以揭示新的可能的BRD4功能途径。 对BRD4活动作用机制的深入了解肯定有助于为所谓的BRD4依赖型癌症开发新的治疗策略。
【背景】表观遗传学领域的研究最近突出了BRD4在癌症进展中的中心作用。 BRD4是BET(溴结构域和终末外结构域)家族(Dey等人,2003; Filippakopoulos等人,2012; Wang)的乙酰赖氨酸阅读器, et al。,2012)能够结合启动子和增强子区域上的乙酰化组蛋白(Dey等人,2003; Filippakopoulos等人,2012; Nagarajan等人,,2014)。这种表观遗传学读者的作用机制在于通过招募几种转录因子,辅因子和RNA聚合酶II(RNApol II)来激活基因启动子和增强子,其导致调节,大部分增强某些靶基因的转录。已经描述了BRD4组蛋白模块发挥调控细胞周期进程的关键作用(Dey等人,2003; Wu和Chiang,2007; Yang等人, ...

Measurement of Mitochondrial DNA Release in Response to ER Stress
Author:
Date:
2016-06-20
[Abstract]  Mitochondria house the metabolic machinery for cellular ATP production. The mitochondrial network is sensitive to perturbations (e.g., oxidative stress and pathogen invasion) that can alter membrane potential, thereby compromising function. Healthy mitochondria maintain high membrane potential due to oxidative phosphorylation (Ly et al., 2003). Changes in mitochondrial function or calcium levels can cause depolarization, or a sharp decrease in mitochondrial membrane potential (Bernardi, 2013). Mitochondrial depolarization induces opening of the mitochondrial permeability transition pore (MPTP), which allows release of mitochondrial components like reactive oxygen species (mtROS), mitochondrial DNA (mtDNA) or intermembrane space proteins into the cytosol ... [摘要]  线粒体代表细胞ATP生产的代谢机制。线粒体网络对可以改变膜电位,从而损害功能的扰动(氧化应激和病原体侵入)敏感。健康的线粒体由于氧化磷酸化维持高的膜电位(Ly等人,2003)。线粒体功能或钙水平的变化可导致去极化,或线粒体膜电位的急剧下降(Bernardi,2013)。线粒体去极化诱导线粒体通透性转换孔(MPTP)的开放,其允许线粒体组分如活性氧(mtROS),线粒体DNA(mtDNA)或膜间隙蛋白释放到细胞质中(Martinou和Green,2001; Tait和Green ,2010; Bronner和O'Riordan,2014)。这些内容触发炎症,并且可导致细胞死亡(West等人,2011)。 mtROS和细胞溶质mtDNA有助于炎症细胞的活化,多蛋白复合物,其处理促炎细胞因子,IL-18和IL-1β。研究表明,胞质mtDNA尤其可以结合两种不同的炎症小体传感器AIM2和NLRP3,导致炎症小体激活(Burckstummer等人,2009; Hornung和Latz,2010)。在此协议中,您将能够特异性提取细胞质mtDNA并使用qPCR测定法定量。


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