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Phosphate buffered salineEither bought as commercially available as filtered sterile phosphate buffered saline (PBS) pH 7.4

磷酸盐缓冲盐水

Company: Sigma-Aldrich
Catalog#: P5493
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Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM)
Author:
Date:
2017-05-05
[Abstract]  To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging secondary ion mass spectrometry (NanoSIMS) generates single cell isotopic composition measurements, and can be used to quantify incorporation and exchange of an isotopically labeled substrate among individual organisms. Here, incorporation of cyanobacterial extracellular organic matter (EOM) by members of a cyanobacterial mixed species biofilm is used as a model to illustrate this method. Incorporation of stable isotope labeled (15N and 13 ... [摘要]  为了提高对微生物相互作用的理解,解决复杂群落中微生物代谢贡献的重要性越来越重要。生物膜的生物体特别难以分离,形成和分析,需要解决这些局限性的方法。高分辨率成像二次离子质谱(NanoSIMS)产生单细胞同位素组成测量,可用于量化在各生物体内同位素标记的底物的掺入和交换。在这里,使用由蓝藻混合物种生物膜的成员掺入的蓝细菌胞外有机物(EOM)作为模型来说明该方法。通过两组(蓝细菌和相关的异养微生物)掺入稳定同位素( 15 N和 13 C)EOM的量化。描述了用于定量生物膜中稳定同位素标记的EOM摄取的样品的生成,制备和分析方法。

背景 与NanoSIMS(“NanoSIP”)结合的稳定同位素标记是一种确定的稳定同位素标记底物掺入到单个微生物细胞中的方法,然后可将其推断以估计细胞群体的掺入(例如,Lechene et al。 ,2006和Woebken等人,2012)。可以将多个稳定同位素标记(例如, 13 C和 15 ...

Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes
Author:
Date:
2017-05-05
[Abstract]  Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like cervical, liver, dendritic, intestinal and macrophages have been used to study in vitro propagation and survival of listeria in human cells. Human intestinal epithelial cells have been used to study how listeria crosses the intestinal barrier and cause infection. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. During adhesion assay the cells are ... [摘要]  单核细胞增生利斯特氏菌是一种重要的革兰氏阳性食源性病原体,是即食食品中的一个特殊问题。它具有在诸如制冷温度和高盐浓度的恶劣条件下生存的能力,并且已知可以穿过肠,胎盘和血脑屏障。已经使用了诸如子宫颈,肝脏,树突状细胞,肠和巨噬细胞的几种癌细胞系来研究人细胞中李斯特菌的体外扩增和存活。人肠上皮细胞已被用于研究李斯特菌如何穿过肠屏障并引起感染。本文中的方案描述了生长Caco-2细胞的过程,维持细胞并将其用于粘附和侵袭测定。在粘附测定期间,将细胞与李斯特菌孵育30分钟,但是在侵袭测定中,细胞生长在感染后的几个时间点被停止以监测细胞中李斯特菌的生长和存活率。

背景 ...

In vitro Microtubule Bundling Assay under Physiological Conditions
Author:
Date:
2017-04-05
[Abstract]  Kinesins play a role in organizing the mitotic spindle through the crosslinking of microtubules (MTs), made possible through binding sites at opposite ends of the holoenzyme. Here, we developed a method to test kinesin MT crosslinking action under physiological conditions. [摘要]  驱动蛋白通过微管(MT)的交联来组织有丝分裂纺锤体,通过在全酶的相对端的结合位点成为可能。在这里,我们开发了一种在生理条件下测试驱动蛋白MT交联作用的方法。

基于微管的运动蛋白是重要的,因为它们使用ATP的化学能产生力以便向量转移微管(MT)。在这些基于MT的运动蛋白中,称为驱动蛋白的超家族负责定向运输和沿微管的运动。一些运动蛋白在全酶的两端具有MT结合位点,因此它们可以在生理ATP条件下将MT交联成束(Tao等人,2006)。由于这种捆绑活动,它们在组织和维持有丝分裂纺锤体方面具有重要作用,其主要作用取决于其微管的极性模式(van den Wildenberg等人,2008)。以前的捆绑测定不包括ATP,或者使用可以产生人为结果的不可水解的ATP类似物。这里我们开发了一种使用生理ATP条件的方法。通过纯化这些全长运动蛋白,它使我们能够在生理条件下测定其交联活性。

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