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DMEM, high glucose, GlutaMAX(TM), pyruvate

DMEM

Company: Thermo Fisher Scientific
Catalog#: 31966047
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Quantification of Neisseria meningitidis Adherence to Human Epithelial Cells by Colony Counting
Author:
Date:
2018-02-05
[Abstract]  To cause an infection, the human specific pathogen Neisseria meningitides must first colonize the nasopharynx. Upon tight interaction with the mucosal epithelium, N. meningitidis may cross the epithelial cellular barrier, reach the bloodstream and cause sepsis and/or meningitis. Since N. meningitidis niche is restricted to humans the availability of relevant animal models to study host-pathogen interactions are limiting. Therefore, most findings that involve N. meningitidis colonization derive from studies using cultured human cell lines. Human epithelial cells have been successfully used to examine and identify molecular effectors involved in initial adherence of the pathogen. Here, we describe a standard protocol to quantify the adherence of N. ... [摘要]  为了引起感染,人类特定的病原体脑膜炎奈瑟氏脑必须首先在鼻咽中定居。 与粘膜上皮紧密相互作用时, 脑膜炎双球菌可能穿过上皮细胞屏障,到达血流并引起败血症和/或脑膜炎。 由于 N meningitidis niche限于人类。 因此,大多数涉及N 脑膜炎菌群定植来源于使用培养的人类细胞系的研究。 在最初的病原体坚持。 在这里,我们描述了一个标准协议来量化N的遵守情况。 脑膜炎双球菌对上皮细胞FaDu细胞的作用。 感染后收集的细胞裂解物的集落计数用于量化对上皮细胞的粘附。

【背景】作为细菌发病机制的重要一步。细菌粘附素与宿主细胞表面受体之间的分子相互作用决定了定植位点(Soto and Hultgren,1999)。鼻咽中的上皮层形成人类限制性病原体N的第一个细胞屏障。 meningitidis 遇到和无症状殖民。牢固粘附,并与宿主细胞相互作用可导致上皮细胞和进入血液的渗透,在危及生命的败血症和/或脑膜炎(斯蒂芬斯,2009)得到的。从细菌膜延伸出来的长细丝(称为IV型菌毛(Tfp))与PilC1尖端定位的粘附素在初始依从性中起关键作用。脑膜炎到鼻咽上皮细胞(Marceau等,1995; Rudel等,1995)。 Tfp不仅促进与宿主细胞的相互作用,而且参与细菌聚集体的发育,这可以有助于高水平的粘附和抵抗剪切应力(Helaine等,2005,Mikaty ...

Relative Stiffness Measurements of Tumour Tissues by Shear Rheology
Author:
Date:
2017-05-05
[Abstract]  The microenvironment of solid tumours is a critical contributor to the progression of tumours and offers a promising target for therapeutic intervention (Cox and Erler, 2011; Barker et al., 2012; Cox et al., 2016; Cox and Erler, 2016). The properties of the tumour microenvironment vary significantly from that of the original tissue in both biochemistry and biomechanics. At present, the complex interplay between the biomechanical properties of the microenvironment and tumour cell phenotype is under intense investigation. The ability to measure the biomechanical properties of tumour samples from cancer models will increase our understanding of their importance in solid tumour biology. Here we report a simple method to measure the viscoelastic properties of tumour specimens ... [摘要]  实体肿瘤的微环境是肿瘤进展的关键因素,为治疗干预提供了有希望的靶点(Cox和Erler,2011; Barker等人,2012; Cox等人,2016; Cox和Erler,2016)。肿瘤微环境的特性与生物化学和生物力学中原始组织的性质差异显着。目前,微环境生物力学特性与肿瘤细胞表型之间的复杂相互作用正在进行深入的研究。从癌症模型中测量肿瘤样本的生物力学性质的能力将增强我们对于在实体肿瘤生物学中的重要性的理解。在这里,我们报告一种使用受控应变旋转流变仪测量肿瘤标本的粘弹性的简单方法。

背景 实体瘤的生长伴随着天然组织的病理重塑(Cox和Erler,2011; Bonnans等人,2014)。在进行期间,局部组织环境经历物理和生物变化,导致组织刚度(弹性模量)增加(Humphrey等人,2014)。细胞外基质的改变导致新的组织特性的产生,其激活肿瘤细胞内的机械信号通路(DuFort等人,2011)。这种外部信号传导导致行为改变,细胞形态,分化,增殖,迁移和干性。在癌症的临床前动物模型中,这些变化已被证明可以驱动恶性进展和转移性扩散(Erler等人,2006; Levental等人,2009; Bonnans 等,,2014)。因此,结果,近年来,基质重塑,特别是硬化的靶向受到了极大的关注,并且已经开始了若干临床试验(Barker等人,2012; ...

Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells
Author:
Date:
2017-04-20
[Abstract]  The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from HEK-293 cells, making use of inducible ectopic expression, affinity capture, and rate-zonal centrifugation. The obtained RNA exosomes have been used successfully for proteomic, structural, and enzymatic studies (Domanski et al., 2016). [摘要]  RNA外植体复合物在RNA加工和调节营养中起核心作用。在细胞质和细胞核中存在,外来体通过与核糖核酸酶和各种衔接蛋白的关联起作用(参见[Kilchert等人,2016])。以下方案描述了从HEK-293细胞中纯化RNA外来体复合物的方法,利用可诱导的异位表达,亲和力捕获和速率 - 区带离心。所获得的RNA外来体已被成功地用于蛋白质组学,结构和酶学研究(Domanski等人,2016)。

在我们以前的工作中,我们建立了在四环素诱导型CMV启动子(HEK-293 Flp-In T-REx-Thermo Fisher)的控制下表达C末端3xFLAG标记的外来体组分EXOSC10(RRP6)的同基因HEK-293细胞系科学)。该系统允许我们以与内源WT蛋白质相当的水平表达标记的EXOSC10蛋白质,并且使用磁性抗FLAG亲和介质和来源于冷冻细胞粉末的蛋白质提取物来研究外来体纯化方案(Domanski等, / em>。,2012)。进一步探索使用的蛋白质提取条件,我们开发了一种允许在亲和捕获的外来体内保留DIS3(RRP44)的方案,否则证明是困难的(Hakhverdyan等人,2015)。基于这些研究,我们通过使用甘油密度梯度的速率区带离心法进一步纯化了RNA外来体(+/- DIS3)(Domanski等人,2016)。虽然蛋白质提取物中洗涤剂CHAPS(3 - ...

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