Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice
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Author:
Date:
2020-05-20
[Abstract] Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for understanding how innate immune cells respond to infection but is technically challenging due to the small number of bacteria that initially infect the lung. Previous studies either evaluated Mtb-infected cells at later stages of infection when the number of ...
[摘要] [摘要 ] 结核分枝杆菌(Mtb)通过气溶胶传播,可引起严重的肺部细菌感染,如果不及时治疗,可能致命。Mtb现在已成为全球传染病致死的主要原因。表征气溶胶激发后体内感染的早期事件对于了解先天免疫细胞如何对感染做出反应至关重要,但由于最初会感染肺的细菌数量少,因此在技术上具有挑战性。先前的研究或者在肺部细菌数量高得多时在感染后期评估Mtb感染的细胞,或者在体外使用 评估骨髓细胞对Mtb反应的模型系统。在这里,我们介绍一种使用荧光细菌,大剂量气溶胶感染模型和流式细胞术跟踪气溶胶感染和荧光激活细胞分选(FACS)之后立即分离肺中Mtb感染细胞的方法,以分离幼稚的旁观者,和Mtb感染的细胞用于下游应用,包括RNA测序。该协议提供了在肺环境中监视Mtb感染和细胞特异性反应的能力,已知该环境可调节常驻和募集人群的功能。使用此协议,我们发现肺泡巨噬细胞通过上调受转录因子Nrf2调节并有害于细菌早期控制的细胞保护性转录反应,在体内对Mtb感染作出反应。
[背景 ] 气溶胶传播是结核分枝杆菌(Mtb)感染自然周期的关键组成部分,有助于细菌的毒性并导致其在肺部的独特感染模式(North ,1995;Riley 等,1995)。 ; Pai et ...
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Vascular Permeability Assay in Human Coronary and Mouse Brachiocephalic Arteries
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Author:
Liang Guo, Raquel Fernandez, Atsushi Sakamoto, Anne Cornelissen, Ka Hyun Paek, Parker J. Lee, Leah M. Weinstein, Carlos J. Collado-Rivera, Emanuel Harari, Robert Kutys, Torie S. Samuda, Nicole A. Singer, Matthew D. Kutyna, Frank D. Kolodgie, Renu Virmani and Aloke V. Finn,
Date:
2018-10-20
[Abstract] Coronary artery disease remains an important cause of morbidity and mortality. Previous work, including ours, has focused on the role of intraplaque hemorrhage, particularly from immature microvessel angiogenesis, as an important contributor to plaque progression via increases in vascular permeability leading to further intraplaque hemorrhage, which increases red cell membrane-derived free cholesterol in plaque content and inflammatory cell recruitment. Evans Blue Dye (EBD) assay is widely used as a standard assay for vasculature permeability. However, the method has not been established in ...
[摘要] 冠状动脉疾病仍然是发病率和死亡率的重要原因。以前的研究,包括我们的研究,都集中在斑块内出血的作用,特别是来自未成熟的微血管血管生成,作为斑块进展的重要因素,通过增加血管通透性导致进一步的斑块内出血,增加斑块中红细胞膜来源的游离胆固醇内容和炎症细胞募集。 Evans Blue Dye(EBD)测定法广泛用作脉管系统渗透性的标准测定法。然而,该方法尚未在新鲜人冠状动脉尸检样本中建立,以评估斑块内微血管通透性和血管生成。在该方案中,我们描述了评估人类冠状动脉样本的微血管通透性的方法,包括灌注冠状动脉的程序,用于组织学分析和免疫染色的动脉样本的收集以及使用适当的方法来分析图像。还提供了在小鼠模型中使用FITC-葡聚糖以评估血管通透性的任选程序。这些Evans Blue Dye程序可用于在各种病理条件下提供人样品和动物模型中内皮完整性和渗透性的功能测量。
【背景】 血管内皮细胞主动调节血浆成分和循环细胞(包括白细胞)从血液到亚内皮组织的浸润。这种机制通常被认为是动脉粥样硬化起始和发展的关键步骤(Mundi et al。>,2018)。血管通透性的调节通过内皮细胞 - 细胞连接的协调打开和闭合来实现。在几种疾病状态下,内源性药物如组胺,凝血酶和血管内皮生长因子(VEGF)显着但可逆地以不同方式改变细胞 - ...
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Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System
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Author:
Date:
2017-10-20
[Abstract] A memory trace, also known as a memory engram, is theorized to be a mechanism for physical memory storage in the brain (Silva et al., 2009; Josselyn, 2010) and memory trace is associated with a specific population of neurons (Liu et al., 2012; Ramirez et al., 2013). Labeling and stimulating those neurons will activate the memory trace (Liu et al., 2012; Ramirez et al., 2013). Memory appears to be spread over different regions of the brain rather than being localized to one area. Therefore, the methods used to trace memory have the ability to ...
[摘要] 存储器跟踪(也称为存储器枚举)被理论化为大脑中物理存储器存储的机制(Silva等人,2009; Josselyn,2010),并且内存跟踪与一个 特定的神经元群体(Liu et al。,2012; Ramirez等人,2013)。 标记和刺激那些神经元将激活记忆痕迹(Liu et al。,2012; Ramirez等人,2013)。 记忆似乎分布在大脑的不同区域,而不是局限于一个区域。 因此,用于跟踪记忆的方法有能力提高我们对神经元电路的理解。 在本协议中,我们引入多西环素诱导表达系统来标记与原始记忆痕迹相关的特定神经元。 【背景】记忆痕迹是记忆被存储为大脑物理或生物化学变化的理论手段(Ryan等人,2015)。在二十世纪初德国动物学家理查德·塞蒙(Richard Semon)制定记忆追踪概念之后,记忆存储的具体过程一直是神经科学领域辩论的一个未解决的话题(Poo et al。,2016)。尽管记忆机制已经成为几十年来的争论焦点,但已经一致认为,特定的神经元被用于记忆的存储(Liu等人,2012; Ramirez等人, ...
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