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DPBS, no calcium, no magnesium

DPBS,无钙,无镁

Company: Thermo Fisher Scientific
Catalog#: 14190250
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Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy
Author:
Date:
2018-06-20
[Abstract]  Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition receptors (PRRs) capable of detecting viral dsRNAs such as endosomal toll-like receptor 3 (TLR3) and cytosolic RIG-I-like receptors (RLRs) which lead to the production of type I interferons (IFNs). Thus, intracellular localization of viral dsRNA can provide insight into the downstream signaling pathways leading to innate immune activation. Here, we describe a quantitative method for measuring extracellular dsRNA uptake and visualizing subcellular ... [摘要]  双链RNA是一种有效的病原体相关分子模式(PAMP),作为病毒复制的副产物和病毒感染的众所周知的标志产生。 病毒dsRNA可以从感染的细胞释放到细胞外空间并通过胞吞作用被邻近细胞内化。 哺乳动物具有能够检测导致产生I型干扰素(IFN)的病毒dsRNA(例如内体Toll样受体3(TLR3)和胞质RIG-1样受体(RLR))的多模式识别受体(PRR)。 因此,病毒dsRNA的细胞内定位可以提供对导致先天免疫激活的下游信号传导途径的了解。 在这里,我们描述了一种测量细胞外dsRNA摄取和分别通过流式细胞仪和共聚焦显微镜观察内化dsRNA的亚细胞定位的定量方法。

【背景】双链RNA(dsRNA)是病毒复制的常见副产物,通过产生I型干扰素(IFN)和其他促炎细胞因子(Nellimarla和Mossman,2014)是抗病毒免疫的有效激活剂。病毒的dsRNA通过TLR3核内体内所感测(松本等人,2003年)或在由RIG-I样受体(RLRS),RIG-I和MDA-5(加藤等人,2006)。在裂解感染,这些dsRNA可以被释放到细胞外空间,它们结合于相邻小区,如A类清道夫受体(SR-A)和RAFTLIN表面受体,并且随后经由网格蛋白介导的内吞作用内在化(伊藤制 2008; DeWitte-Orr等人,2010; Watanabe等人,2011; Dansako等人,, ...

Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism
Author:
Date:
2018-05-20
[Abstract]  Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs (Koppenol et al., 2011; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza et al., 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements ... [摘要]  哺乳动物细胞通过线粒体(氧化磷酸化)和非线粒体(糖酵解)代谢产生ATP。已知癌细胞使用不同的策略重新编程它们的代谢以满足能量和合成代谢需要(Koppenol等人,2011; Zheng,2012)。此外,每个癌症组织都有其自己的个体代谢特征。线粒体不仅在能量代谢中起关键作用,而且在细胞的细胞周期调控中也起关键作用。因此,线粒体作为抗癌治疗的潜在靶标已经出现,因为它们在结构和功能上与其非癌对应物不同(D'Souza等人,2011)。我们详细介绍了利用海马XF24细胞外通量分析仪(图1)测量活细胞中氧耗率(OCR)和细胞外酸化率(ECAR)测量的方案。 Seahorse XF24细胞外通量分析仪持续测量细胞上清液中的氧浓度和质子流量(Wu等人,2007)。这些测量结果在OCR和ECAR值中转换,并能够直接定量线粒体呼吸和糖酵解。有了这个协议,我们试图评估三种不同癌细胞系的基线粒体功能和线粒体应激反应细胞毒性测试先导化合物甲磺卡西林,以研究其作用机制。将细胞铺在XF24细胞培养板中并保持24小时。在分析之前,将培养基替换为无缓冲的DMEM pH7.4,然后使细胞在非代谢通量分析前使用Seahorse XF在非CO 2孵育器中平衡以允许精确测量Milli-pH单位改变。 ...

In vitro Co-culture of Mesenchymal Stem Cells and Endothelial Colony Forming Cells
Author:
Date:
2017-10-20
[Abstract]  The discovery of endothelial colony forming cells (ECFCs) with robust self-renewal and de novo vessel formation potentials suggests that ECFCs can be an excellent cell source for cardiovascular diseases treatment through improving neovascularization in the ischemic tissues. However, their engraftment after transplantation resulted to be low. Previous studies showed mesenchymal stem/stromal cells (MSCs) could improve the survival and capillary formation capacity of ECFCs in co-culture systems. In this article, we describe a protocol for in vitro co-culture of MSCs and ECFCs to prime ECFCs for better engraftment. [摘要]  发现具有强大自我更新和从头血管形成潜力的内皮细胞集落形成细胞(ECFCs)表明,ECFC可以通过改善缺血组织的新生血管形成,成为心血管疾病治疗的优良细胞来源。 然而,移植后的移植导致了低位移植。 以前的研究显示间充质干/基质细胞(MSC)可以改善共培养系统中ECFCs的存活和毛细管形成能力。 在这篇文章中,我们描述了体外协调MSCs和ECFCs共同培养ECFC以实现更好的移植。
【背景】内皮祖细胞(EPC)被定义为能够通过血管发生过程形成新血管的细胞群。 2004年,Ingram等人鉴定了来自人脐带血的称为“内皮细胞集落形成细胞(ECFC)”的离体培养物中的特异性高度增殖的EPC群体Ingram等人,2004),并且这些细胞最近被宣布代表EPCs(Medina等人,2017)。类似的群体也可以从具有等效血管化潜力和临床相关数量的人类胎盘组织中分离(Patel等人,2013; Shafiee等人,2015) )。因此,ECFC移植已被提出作为缺血性疾病如心肌梗塞或关键性腿部缺血的治疗方法。然而,移植后的ECFCs植入物和血管生成潜力被证明是低的(Shafiee等人,2017; ...

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