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HBSS

HBSS,无钙,无镁

Company: Thermo Fisher Scientific
Catalog#: 14170112
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Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
Author:
Date:
2018-07-20
[Abstract]  After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow ... [摘要]  在识别细胞外细菌脂多糖(LPS)后,Toll样受体4(TLR4)-CD14信号传导复合物启动两种不同的信号传导途径 - 一种来自质膜,另一种来自信号传导内体(Kagan 等。,2008)。因此,了解TLR4信号转导的早期阶段需要一种稳健且定量的方法来测量LPS触发的TLR4和CD14受体内吞作用,这是LPS检测中最早发生的事件之一。在这里,我们描述了一种基于流式细胞术的方法,我们最近用它来研究离子通道TRPM7在TLR4内吞作用中的作用(Schappe et al。,2018)。该测定依赖于用LPS刺激细胞并使用流式细胞术在不同时间点测量TLR4(或CD14)的细胞表面水平。尽管我们详细描述了来自鼠骨髓来源的巨噬细胞的TLR4和CD14的方法,但它可以很容易地适应于在各种其他信号传导环境中评估受体内吞作用。

【背景】先天免疫细胞,包括巨噬细胞和树突细胞,使用各种模式识别受体(PRR)来调查其环境中的危险和病原体相关分子模式。来自各种亚细胞区室的PRR的贩运和信号传导实现了更广泛的免疫监视,并且已成为先天免疫的重要设计原则(Brubaker et al。,2015)。细菌内毒素LPS的检测高度依赖于TLR4及其共同受体CD14。 TLR4复合物的内吞作用需要CD14,并且对于LPS诱导的巨噬细胞活化是必需的(Zanoni 等人,2011; Tan ...

Isolation and Separation of Epithelial CD34+ Cancer Stem Cells from Tgfbr2-deficient Squamous Cell Carcinoma
Author:
Date:
2017-09-05
[Abstract]  Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further ... [摘要]  大多数上皮肿瘤已经显示含有可能是肿瘤进展和转移的驱动力的癌症干细胞(Kreso和Dick,2014; Nassar和Blanpain,2016)。 为了深入研究这些细胞,依赖于细胞表面标志物或荧光报告基因的细胞分离策略是必不可少的,分离策略必须保持其活力。 从大部分肿瘤中分离不同细胞群的能力将继续加深我们对癌症干细胞生物学的认识。 在这里,我们报告了结合机械肿瘤解离,酶处理和流式细胞术的策略,从其天然微环境中分离出纯种群的上皮癌干细胞。 该技术可用于进一步功能性地分析癌症干细胞(RNA测序和表观遗传学分析),在培养物中培养它们或在移植测定中直接使用它们。
【背景】肿瘤复发和转移是大多数与癌症有关的死亡的主要原因。恶性肿瘤可能由干细胞群体启动和维持(Nassar和Blanpain,2016; Bonnet和Dick,1997),这些细胞是预防复发的重要治疗靶点(Baumann et al。,2008)。研究表明,鳞状细胞癌由肿瘤干细胞亚群维持,其抗药性,并通过进行自我更新和分化(如正常干细胞)引发肿瘤复发,产生增殖祖细胞,其分化形成肿瘤大部分(Locke et al。,2005; Prince et al。,2007; Malanchi et al。,2008; de Sousa e Melo et ...

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis
Author:
Date:
2017-07-20
[Abstract]  Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated. These alginate-based matrices produce highly reproducible results and can be mixed with different extracellular matrix components. This protocol ... [摘要]  标准细胞培养模型已被用于调查疾病病理学和测试新疗法超过五十年。 然而,这些模型系统通常未能模拟在体内发生病理学的三维(3-D)空间内发生的变化。 为了真实地表示这一点,生物学中新兴的范例是在生理相关的三维环境中建模疾病的重要性。 3-D细胞培养的方法之一是生物电喷雾技术。 该技术使用基于藻酸盐的3-D环境作为惰性骨架,其中可以并入哺乳动物细胞和细胞外基质。 这些基于藻酸盐的基质产生高度可重复的结果,并且可以与不同的细胞外基质组分混合。 该方案描述了一种结合分枝杆菌,原代人血液单核细胞和胶原 - 藻酸盐基质的3-D系统,以解剖结核病中宿主病原体的相互作用。
【背景】结核分枝杆菌(Mtb)是全球公共卫生重要性的病原体,每年造成180万人死亡,全球发病率达到1000万(世卫组织,2016年)。尽管在研究方面进行了大量投资,但需要更多地了解宿主 - 病原体的相互作用才能改善预防和治疗。目前,病原菌对常用药物的耐药性越来越高,出现广泛耐药Mtb。结核病(TB)领域的挑战之一是可用于询问宿主 - 病原体相互作用的模型系统,因为广泛使用的动物模型不能完全反映人类的病理学。因此,迫切需要通过开发与生理相关的体外环境(Bielecka等人,2017年; Tezera等人)来补充这些动物模型。,2017)。 ...

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