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Potassium chloride


Company: Sigma-Aldrich
Catalog#: P9541
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Protocol for the Isolation and Super-resolution dSTORM Imaging of RyR2 in Cardiac Myocytes
[Abstract]  Since its inception, super-resolution microscopy has played an increasingly important role in the discovery and characterization of nanoscale biological structure. dSTORM, which is one of the most commonly applied methods, relies on stochastic photoswitching of fluorophores to recreate a super-resolution image. The cardiac field has particularly benefitted from the application of this technique, as it has enabled sub-diffraction-limit visualization of calcium release units (CRUs) and the fundamental structures that trigger contraction. Acquisition of such images requires careful, reproducible sample preparation, and consistent imaging conditions maintained for the duration of the experiment. Here we present standardized methods for the production of dSTORM images of the Ca2+ ... [摘要]  自成立以来,超分辨率显微镜在纳米级生物结构的发现和表征中发挥着越来越重要的作用。 dSTORM是最常用的方法之一,它依赖于荧光团的随机光切换来重建超分辨率图像。心脏场特别受益于该技术的应用,因为它已经实现了钙释放单元(CRU)的子衍射极限可视化和触发收缩的基本结构。获取这些图像需要仔细,可重复的样品制备,并且在实验期间保持一致的成像条件。在这里,我们提出了生产心肌细胞中Ca 2 + 释放通道Ryanodine Receptor type-2(RyR2)的dSTORM图像的标准化方法。所提出的方案特别关注涉及原发性心肌细胞分离,样品制备和成像的步骤,其中提供了针对实验溶液和显微镜设置的细节。本讨论之后是各种分析技术的概述,以识别集群和CRU中的RyR2组织

【背景】近年来,超分辨率显微镜的普及率迅速提高。已经描述了各种超分辨率技术,其使光学分辨率远低于光的衍射极限,在某些情况下接近可通过电子显微镜获得的光学分辨率。总之,这些技术的出现导致了纳米级生物结构,结构域和蛋白质相互作用的新研究的爆炸式增长。一种流行的超分辨率技术是直接随机光学显微镜(dSTORM),与标准共聚焦显微镜相比,它将相对简单的样品处理的优势与分辨率提高了约10倍(van de Linde ...

Bacterial Microcolonies in Gel Beads for High-throughput Screening
[Abstract]  High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants.

Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies ...
[摘要]  在细菌群体中表达的DNA文库的高通量筛选用于鉴定显示感兴趣性质的潜在稀有成员是在许多实验中成功的关键步骤,例如蛋白质和合成回路的定向进化以及用于鉴定增益的深度突变扫描 - 或功能丧失的突变体。

在这里,我描述了一种用于高通量筛选凝胶珠中细菌(大肠杆菌)微菌落的方案。将单细胞包封成用微流体装置产生的单分散油包水乳液液滴。水溶液还含有琼脂糖,其在冰上冷却时凝胶化,从而在液滴内部形成固体凝胶珠。在乳液温育期间,细胞在珠内生长成单克隆微菌落。在从乳液中分离凝胶珠并通过荧光激活细胞分选(FACS)分选后,从凝胶珠中回收细菌,然后准备进行进一步的分选,诱变或分析。为了通过FACS分类,该方案需要荧光读数,例如荧光报告蛋白的表达。测量微小菌落的平均荧光信号降低了高表型细胞间变异性的影响,并且与单细胞分选相比提高了灵敏度。我们应用这种方法在ON和OFF状态下对pBAD启动子文库进行分类(Duarte et al。,2017)。

【背景】荧光激活细胞分选(FACS)具有> 10 7 事件/ h的无与伦比的筛选通量(Davies,2012)。然而,通过FACS根据其荧光分选单个细胞以筛选合成回路的文库(Schaerli和Isalan,2013)经常受到高表型细胞间变异性的阻碍。或者,可以对水凝胶珠中所含的小细胞集落(微集落)进行分类(Weaver ...

Sacral Spinal Cord Transection and Isolated Sacral Cord Preparation to Study Chronic Spinal Cord Injury in Adult Mice
[Abstract]  Spinal cord injury (SCI) is characterized by multiple sensory/motor impairments that arise from different underlying neural mechanisms. Linking specific sensory/motor impairments to neural mechanism is limited by a lack of direct experimental access to these neural circuits. Here, we describe an experimental model which addresses this shortcoming. We generated a mouse model of chronic spinal cord injury that reliably reproduces spasticity observed after SCI, while at the same time allows study of motor impairments in vivo and in an in vitro preparation of the spinal cord. The model allows for the combination of mouse genetics in in vitro and in vivo conditions with advanced imaging, behavioral analysis, and detailed electrophysiology, techniques which ... [摘要]  脊髓损伤(SCI)的特点是由不同的潜在神经机制引起的多种感觉/运动损伤。 将特定的感觉/运动障碍与神经机制联系起来受限于缺乏对这些神经回路的直接实验访问。 在这里,我们描述了一个解决这个缺点的实验模型。 我们产生了一种慢性脊髓损伤的小鼠模型,其可靠地再现SCI后观察到的痉挛状态,同时允许研究运动损伤体内和体外制剂 的脊髓。 该模型允许将体外和体内条件下的小鼠遗传与高级成像,行为分析和详细的电生理学技术相结合,这些技术在常规SCI中不易应用 楷模。

【背景】脊髓损伤导致了毁灭性的感官 - ...