| Brain-localized and Intravenous Microinjections in the Larval Zebrafish to Assess Innate Immune Response
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Author:
Date:
2021-04-05
[Abstract] Creating a robust and controlled infection model is imperative for studying the innate immune response. Leveraging the particular strengths of the zebrafish model system, such as optical transparency, ex utero development, and large clutch size, allows for the development of methods that yield consistent and reproducible results. We created a robust model for activation of innate immunity by microinjecting bacterial particles or live bacteria into larval zebrafish, unlike previous studies which largely restricted such manipulations to embryonic stages of zebrafish. The ability to introduce stimuli locally or systemically at larval stages provides significant advantages to examine host response in more mature tissues as well as the possibility to interrogate adaptive immunity at older ...
[摘要] [摘要]创建一个健壮和控制感染模型是势在必行用于学习的先天免疫应答。凭借特殊的斑马鱼模型系统的优势，比如光学透明性，前子宫发展，以及大离合器的尺寸，允许小号的了的，其产生方法的发展是一致和可重复的 结果。我们通过将细菌颗粒或活细菌显微注射到幼虫斑马鱼中创建了一个强大的激活先天免疫的模型，这与以前的研究在很大程度上将此类操作限制在斑马鱼的胚胎阶段不同。在幼虫阶段局部或全身引入刺激的能力提供了显着的优势，可检查更成熟组织中的宿主反应，并在较老的幼虫阶段询问适应性免疫的可能性。这个协议描述2种显微注射的不同模式以引入脂多糖（LPS）或细菌到活幼虫斑马鱼：一个本地化到的脑，和另一个为对经由尾静脉血流丛。
图形摘要：
示意性示出了两种不同的模式的幼虫斑马鱼显微注射，无论是在脑实质或在血流中静脉内。ř eagents引入斑马鱼，以评估免疫应答中作为在协议中描述的“注入组件”被描绘。
[背景]感染过程中复杂的相互作用需要使用体内动物模型来充分了解病原体与其宿主之间的动态相互作用。研究此现象需要病原体传递的受控和可靠方法。斑马鱼已被用作研究多种病原体免疫应答的模型（Menudier等，1996; Davis等，2002; Neely等，2002; Prouty等，2003; van der ...
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| Real-time in vivo Imaging of LPS-induced Local Inflammation and Drug Deposition in NF-κB Reporter Mice
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Author:
Date:
2020-08-20
[Abstract] Wound, biomaterial, and surgical infections are all characterized by a localized and excessive inflammation, motivating the development of in vivo methods focused on the analysis of local immune events. However, current inflammation models, such as the commonly used in vivo models of endotoxin-induced inflammation are based on systemic, usually intraperitoneal, administration of lipopolysaccharide (LPS), causing endotoxin shock. Here, we describe a model of LPS-induced local inflammation in NF-κB-RE-Luc reporter mice. LPS, alone or with added therapeutic substances, is delivered locally via a hydrogel which is deposited subcutaneously, providing a spatially defined environment, enabling in vivo bioimaging analyses of local NF-κB activation. Evaluation of drug ...
[摘要] [摘要 ] 伤口，生物材料和外科手术感染均以局部和过度炎症为特征，从而推动了专注于分析局部免疫事件的体内方法的发展。然而，当前的炎症模型，例如内毒素诱导的炎症的常用体内模型，是基于系统性的，通常是腹膜内注射脂多糖（LPS），引起内毒素休克。在这里，我们描述了LPS诱导局部炎症的模型NF- κ 乙-RE-Luc报告小鼠。LPS单独或与其他治疗物质一起通过水凝胶局部递送， 沉积皮下，提供一个空间限定的环境中，使得能够在体内生物成像本地NF-的分析κ 乙活化。可以在同一只小鼠中纵向分析药物功效的评估，并且使用荧光标记的药物，可以同时分析局部药物沉积，并将其与炎症部位相关联。最后，该方案还可以用于研究药物从局部沉积的凝胶和其他生物材料的保留和系统释放。
[背景 ] 局部过度的TLR应答的原因，有时不相称的炎症，如在不同类型的伤口和生物材料感染的观察。这些伤口并发症延迟了正常的愈合，并增加了严重感染和败血症的风险。考虑到后者，已经开发了败血症和内毒素休克的几种实验模型，其研究了系统性炎症的发展（Lewis 等，2016）。然而，需要从机械学和治疗学角度解决局部炎症事件的模型。甲转录ctivation 因子NF- κ 乙是各种炎性病症的关键组成部分，因此，NF- κ 乙被认为是重要的治疗靶标（刘等人，2017） 。实时的，纵向体内NF-的成像κ 乙活化NF κ ...
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| Metabolomic and Lipidomic Analysis of Bone Marrow Derived Macrophages
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Author:
Date:
2020-07-20
[Abstract] Macrophages are highly plastic immune cells that are capable of adopting a wide array of functional phenotypes in response to environmental stimuli. The changes in macrophage function are often supported and regulated by changes in cellular metabolism. Capturing a comprehensive picture of metabolism is vital for understanding the role of metabolic rewiring in the immune response. Here we present a method for systematically quantifying the abundance of metabolites and lipids in primary murine bone marrow derived macrophages (BMDMs). This method simultaneously extracts polar metabolites and lipids from BMDMs using a rapid two-phase extraction procedure. The polar metabolite fraction and lipid fraction are subsequently analyzed by separate liquid chromatography-mass spectrometry (LC-MS) ...
[摘要] [摘要] 巨噬细胞是高度可塑性的免疫细胞，能够对环境刺激产生广泛的功能表型。巨噬细胞功能的改变通常受到细胞代谢的支持和调节。获取新陈代谢的全面图像对于理解代谢重组在免疫反应中的作用至关重要。本文提出了一种系统定量测定小鼠骨髓源性巨噬细胞（BMDMs）中代谢物和脂质丰度的方法。该方法采用快速两相萃取法同时从BMDMs中提取极性代谢物和脂质。极性代谢物部分和脂质部分随后通过单独的液相色谱-质谱（LC-MS）方法进行分析，以优化覆盖率和定量。这使得对细胞代谢的全面表征可以用来理解各种环境刺激对巨噬细胞代谢和功能的影响。
[背景] 巨噬细胞是先天性免疫系统的细胞，在局部微环境中对信号作出反应时，可采用多种功能表型。巨噬细胞的激活与细胞代谢的特定重编程相结合并高度依赖（Tannahill et al.，2013；Galván-Peña and O'Neill，2014；Jha et al.，2015；Kelly and O'Neill，2015；Cordes et al.，2016；Mills and O'Neill 2016；等人；Mills et al.，2016；Liu et al.，2013；Lampropoulou et al.，2016；Van den ...
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