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MgCl2·6H2O
Company: Sigma
Catalog#: M2393
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Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice
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 bacteria in the lung is much higher or used in vitro model systems to assess the response of myeloid cells to Mtb. Here, we describe a method that uses fluorescent bacteria, a high-dose aerosol ... [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 ...

Assessing in vitro and in vivo Trogocytosis By Murine CD4+ T cells
Author:
Date:
2020-05-05
[Abstract]  Recognition of antigens by lymphocytes (B, T, and NK) on the surface of an antigen-presenting cell (APC) leads to lymphocyte activation and the formation of an immunological synapse between the lymphocyte and the APC. At the immunological synapse APC membrane and associated membrane proteins can be transferred to the lymphocyte in a process called trogocytosis. The detection of trogocytosed molecules provides insights to the activation state, antigen specificity, and effector functions and differentiation of the lymphocytes. Here we outline our protocol for identifying trogocytosis-positive CD4+ T cells in vitro and in vivo. In vitro, antigen presenting cells are surface biotinylated and pre-loaded with magnetic polystyrene beads before incubating for ... [摘要]  [摘要 ] 抗原呈递细胞（APC）表面的淋巴细胞（B，T和NK）识别抗原会导致淋巴细胞活化并在淋巴细胞和APC之间形成免疫突触。在免疫突触处APC膜和相关的膜蛋白可以转移调用Trogocytosis到淋巴细胞的过程。检测Trogocytosed分子提供见解的激活状态，抗原特异性和效应器功能和差异的淋巴细胞。这里我们列出了我们的协议，用于识别Trogocytosis CD4阳性Tasu 性T细胞在体外和体内。体外，抗原呈递细胞是表面 生物素化并预装磁性聚苯乙烯珠，然后与体外活化的CD4 + T细胞胚细胞（90分钟）或幼稚T细胞（3-24小时）短时间孵育。阳性（trog + ）细胞可通过链霉亲和素染色来筛选经生物素化的经转钙蛋白的APC膜蛋白，然后立即或在随后的孵育后通过流式细胞仪分析其激活表型，效应子功能和效应子的分化，例如，可以鉴定出嗜光细胞的阳性细胞。以前的研究已经描述了分析T细胞嗜光性的方法，以鉴定抗原特异性细胞或被细胞识别的抗原表位。使用当前方案，嗜光性对单个T细胞的影响或能力trog + T细胞调节其他免疫细胞的激活和功能的能力可在一个扩展范围内进行评估 ed时间段。

[背景 ] Trogocytosis是质膜和膜相关分子的细胞间转移。这种现象已在免疫细胞相互作用的分析中得到了广泛研究，并已观察到包括向CD4 +的转移（Wetzel 等，2005； ...

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