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Trypan blue solution

台盼蓝溶液

Company: Sigma-Aldrich
Catalog#: T8154
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Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-host Interaction
Author:
Date:
2020-11-20
[Abstract]  Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma’s environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the ... [摘要]  [摘要]肉芽肿是有组织的多细胞结构,构成了人类病原体结核分枝杆菌(Mtb )感染的标志。对肉芽肿环境中复杂的宿主-Mtb相互作用的更好理解可能会导致新的治疗或预防工具,以改善对结核病大流行的控制。迄今为止,已经报道了几种能够模仿人类新生肉芽肿的体外模型。在这里我们描述一个协议,其中Mtb 被感染的人外周血单核细胞(PBMC)嵌入胶原蛋白基质中,导致形成三维微肉芽肿。随后,可以检索PBMC和Mtb,从而从宿主和病原体中读取多参数。除了并入生理性细胞外基质外,该模型还具有重现休眠样Mtb特征的独特优势,以及在免疫调节治疗下观察到的Mtb复苏的再生,其他已发表的体外实验方法尚未见报道。肉芽肿。

关键词:分枝杆菌,浴池erculosis,肉芽肿,主机,我ñ体外模型,休眠,复苏



[背景]结核病(TB)是一种空气传播的疾病,其包括由人类病原体肺和肺外感染结核分枝杆菌(Mtb的)。结核病在2019年估计造成150万人死亡(世卫组织,2019年),仍然是世界上最致命的传染病。结核病免疫发病机制的特点是形成了组织性的,称为肉芽肿的多细胞簇(Gengenbacher和Kaufmann,2012)。这些结构主要由被感染的和未感染的巨噬细胞的核心组成,周围是淋巴细胞边缘。肉芽肿内的敌对环境推动Mtb进入可能与疾病的潜在形式相关的缓慢或非复制性休眠状态。因此,Mtb休眠导致对针对分枝杆菌复制过程中活跃的代谢途径的抗生素的耐受性增加。 ...

Confocal Microscopy of Reovirus Transport in Living Dorsal Root Ganglion Neurons
Author:
Date:
2020-11-20
[Abstract]  

Neurotropic reoviruses repurpose host machinery to traffic over long distances in neuronal processes and access distal replication sites. Understanding mechanisms of neuronal transmission is facilitated by using simplified in vitro primary neuronal culture models. Advances in the design of compartmentalized microfluidic devices lend robustness to neuronal culture models by enabling compartmentalization and manipulation of distinct neuronal processes. Here, we describe a streamlined methodology to culture sensory neurons dissociated from dorsal root ganglia of embryonic rats in microfluidic devices. We further describe protocols to exogenously label reovirus and image, track, and analyze transport of single reovirus particles in living neurons. These techniques can be adapted to study

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[摘要]  [摘要] 嗜神经性呼肠孤病毒重新利用宿主机器在神经元过程中进行长距离的传输,并进入远端复制部位。简化的体外原代神经元培养模型有助于理解神经元传递机制。室化微流控装置设计的进展使得不同的神经元过程能够被划分和操作,从而为神经元培养模型提供了稳健性。在这里,我们描述了一种在微流控装置中培养胚胎大鼠背根神经节分离的感觉神经元的方法。我们进一步描述了外源性标记呼肠孤病毒的方法,并对单个呼肠孤病毒粒子在活神经元中的转运进行了成像、跟踪和分析。这些技术可应用于研究其他嗜神经病毒的轴突定向转运以及参与信号传导和病理学的神经因子。



[背景]来自不同家族的病毒,包括黄病毒科、疱疹病毒科、小角RNA病毒科和弹状病毒科,突破神经系统的保护屏障,造成严重的疾病和经济负担(Koyuncu等人,2013年;Bohmwald等人,2018年;Tyler,2018年)。哺乳动物正呼肠孤病毒(reovirus,reovirus)属于呼肠孤病毒科,在多种年轻哺乳动物中引起血清型依赖性神经元感染,可导致致命性脑炎(Tyler等人,1986年;Dermody等人,2013年)。呼肠孤病毒没有被两个同心蛋白壳包裹的片段dsRNA基因组包裹,是研究神经系统病毒感染的一种灵活工具(Dermody等人,2013年)。虽然呼肠孤病毒感染的细胞和分子机制已被广泛地利用转化细胞系进行研究,但这些系统并不能捕捉到极化神经元细胞的复杂性。感染神经元的病毒必须在轴突中长距离传播,才能到达复制和释放的远端。为了了解呼肠孤病毒进入神经元和长距离运输的机制,我们最近采用了一些技术来培养原代神经元,并对活细胞中荧光标记的呼肠孤病毒成像(Aravamudhan等人,2020年)。 ...

Flow Cytometry of CD14, VDR, Cyp27 and Cyp24 and TLR4 in U937 Cells
Author:
Date:
2020-08-05
[Abstract]  Chronic Kidney Disease (CKD) patients present a micro inflammation state due to failure renal function. The calcitriol has been described as an anti-inflammatory factor that might modulates the inflammatory response in CKD patients. However, these patients have deficiency of Calcitriol due to failure renal function. But, synthesis of this vitamin has been reported in extra renal production, as in monocytes. In this context, it has been reported that the supplementation with 25 vitamin D (calcidiol or inactive form of vitamin D) induces monocytes to downregulate inflammation, due to the intracellular 1α-hidroxilase that converts calcidiol to calcitriol in these cells. Besides some reports used RT-qPCR, Western Blot or immunofluorescence techniques to investigate the expression of ... [摘要]  [摘要] 慢性肾脏病(CKD)患者由于肾功能衰竭而出现微炎症状态。骨化三醇被认为是一种抗炎因子,可能调节CKD患者的炎症反应。然而,这些患者由于肾功能衰竭而出现骨化三醇缺乏症。但是,据报道,这种维生素的合成是在肾外产生的,比如在单核细胞中。在这种情况下,有报道称,补充25维生素D(钙二醇或不活跃形式的维生素D)可诱导单核细胞下调炎症反应,这是由于细胞内的1α-羟脯氨酸酶将钙二醇转化为骨化三醇。本研究除采用RT-qPCR、Western-Blot或免疫荧光技术研究炎症和维生素D机械生物标志物在几种疾病中的表达外,本研究还应用流式细胞术技术评价25种维生素D对CD14、Toll样受体4(TLR4)、维生素D受体(VDR)的影响,单核细胞系(U937)中的1-α羟化酶(CYP27)、24-羟化酶(CYP24)。将U937培养物与健康或CKD血清孵育,用或不加25维生素D(50 ng/ml,24 h)处理,以评估CD14、TRL4、VDR、CYP27和CYP24的表达。该方案显示了研究25维生素D处理对细胞内和细胞膜生物标志物表达的影响的优势。此外,与RT-qPCR、westernblot或免疫荧光法相比,该技术不费力,但易于操作和解释。

[背景] ...

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