Isolation and Culture of Mouse Lung ILC2s
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Author:
Date:
2018-10-05
[Abstract] Group 2 Innate Lymphoid Cells (ILC2) play an important role in immune responses at barrier surfaces, notably in the lung during airway allergic inflammation or asthma. Several studies have described methods to isolate ILC2s from wild-type naive mice, most of them using cell sorting to obtain a pure population. Here, we describe in detail, a simple, efficient method for isolation and culture of lung mouse ILC2s. Lungs from Rag2-/- mice pretreated with IL-33 are collected and processed into single cell suspensions. Lymphoid cells are then recovered by density gradient separation. Lin-CD45+ cells are selected by depletion of lineage positive cells followed by positive selection of CD45+ cells. Culture of the isolated cells for several days ...
[摘要] 第2组先天性淋巴细胞(ILC2)在屏障表面,特别是在气道过敏性炎症或哮喘期间的肺中的免疫应答中起重要作用。一些研究已经描述了从野生型幼稚小鼠中分离ILC2的方法,其中大多数使用细胞分选来获得纯种群。在这里,我们详细描述了一种简单有效的肺小鼠ILC2分离和培养方法。收集用IL-33预处理的 Rag2 - / - 小鼠的肺并加工成单细胞悬浮液。然后通过密度梯度分离回收淋巴样细胞。通过耗尽谱系阳性细胞然后阳性选择CD45 + 细胞来选择Lin - CD45 + 细胞。将分离的细胞培养数天导致高度纯化的ILC2群体表达典型的细胞表面标志物(CD90.2,Sca1,CD25,CD127和IL-33R)。这些细胞可在培养物中扩增长达10天,并用于多种离体测定或体内过继转移实验。 【背景】第2组先天性淋巴细胞(ILC2)是组织驻留细胞,其在抗寄生虫先天免疫以及过敏性炎症的发展中起关键作用。它们通过产生大量的2型细胞因子IL-5和IL-13对上皮细胞衍生的细胞因子如白细胞介素-33(IL-33)起反应,后者又诱导嗜酸性粒细胞增多和粘液产生(Cayrol和Girard,2018)。为了更好地表征这些细胞的功能和调节,许多组通过荧光激活细胞分选(FACS)从野生型小鼠(WT)的肺中分选ILC2。由于稳定状态下肺中存在的ILC2数量较少,因此该方法导致纯化细胞的产量较低(每只小鼠1×10 ...
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CRISPR/Cas9-mediated ssDNA Recombineering in Corynebacterium glutamicum
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Author:
Date:
2018-10-05
[Abstract] Corynebacterium glutamicum is a versatile workhorse for industrial bioproduction of many kinds of chemicals and fuels, notably amino acids. Development of advanced genetic engineering tools is urgently demanded for systems metabolic engineering of C. glutamicum. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are now revolutionizing genome editing. The CRISPR/Cas9 system from Streptococcus pyogenes that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum. In this protocol, we described the general procedure for CRISPR/Cas9-mediated ssDNA ...
[摘要] 谷氨酸棒杆菌是多种化学品和燃料,特别是氨基酸的工业生物生产的多功能工具。 迫切需要开发先进的基因工程工具用于 C的系统代谢工程。谷氨酸。 最近推出的聚集的有规律的间隔短回文重复序列(CRISPR)和它们的CRISPR相关蛋白(Cas)现在正在彻底改变基因组编辑。 来自 Streptococcus pyogenes 的CRISPR / Cas9系统利用NGG作为原型间隔区相邻基序(PAM)并具有良好的靶向特异性,可以开发成为 C的高效和精确基因组编辑的有力工具。谷氨酸。 在该方案中,我们描述了 C中CRISPR / Cas9介导的ssDNA重组工程的一般程序。谷氨酸。 可以在 C中引入小的修改。 谷氨酸染色体,编辑效率高达90%。 【背景】革兰氏阳性土壤细菌 Corynebacterium glutamicum 是用于氨基酸,生物燃料和聚合物构建模块的工业生物生产的多功能工具(Becker et al。,2016)。在 C工程的早期阶段。谷氨酸,随机诱变结合对氨基酸类似物的表型抗性的阳性选择是最常用的策略(Vertes et al。,2005)。 C中的遗传操作。谷氨酸(glutamicum)于1984年启动,并成为菌株改良的关键促成策略(Ozaki et al。,1984)。常规使用的基因破坏和插入 ...
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Platelet Migration and Bacterial Trapping Assay under Flow
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Author:
Date:
2018-09-20
[Abstract] Blood platelets are critical for hemostasis and thrombosis, but also play diverse roles during immune responses. We have recently reported that platelets migrate at sites of infection in vitro and in vivo. Importantly, platelets use their ability to migrate to collect and bundle fibrin (ogen)-bound bacteria accomplishing efficient intravascular bacterial trapping. Here, we describe a method that allows analyzing platelet migration in vitro, focusing on their ability to collect bacteria and trap bacteria under flow.
[摘要] 血小板对于止血和血栓形成至关重要,但在免疫反应中也起着不同的作用。 我们最近报道了血小板在体外体外和体内感染部位迁移。 重要的是,血小板利用它们迁移的能力来收集和捆绑纤维蛋白(ogen)结合的细菌,从而实现有效的血管内细菌捕获。 在这里,我们描述了一种方法,允许分析血小板在体外的迁移,重点是它们收集细菌和捕获流动细菌的能力。
【背景】血小板是从巨核细胞释放的小的无核细胞片段,其存在于哺乳动物生物的骨髓内(Machlus和Italiano,2013)。大约7500亿血小板在人体血液中循环,不断扫描脉管系统以破坏内皮表面。在遇到内皮损伤时,血小板立即被招募在充分表征的事件级联中,包括初始血小板束缚和滚动,然后是血小板活化,粘附和扩散,最终导致纤维蛋白(ogen)依赖性聚集和随后的血栓收缩(Jackson, 2007)。血小板栓塞形成是生理性止血的主要步骤,但也是动脉粥样硬化斑块破裂后的病理性血栓形成,触发心肌梗塞或中风(Jackson,2011)。
除了在止血和血栓形成中的公认作用外,血小板还发展出多种免疫功能(Semple et al。,2011)。作为第一批招募炎症和感染部位的细胞,血小板在启动血管内免疫反应中起着重要作用(Wong et ...
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