| 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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| Identification and Quantitation of Leukocyte Populations in Human Kidney Tissue by Multi-parameter Flow Cytometry
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Author:
Date:
2018-08-20
[Abstract] Inflammatory immune cells play direct pathological roles in cases of acute kidney injury (AKI) and chronic kidney disease (CKD). However, the identification and characterization of distinct populations of leukocytes in human kidney biopsies have been confounded by the limitations of immunohistochemical (IHC)-based techniques used to detect them. This methodology is not amenable to the combinations of multiple markers necessary to unequivocally define discrete immune cell populations. We have developed a multi-parameter, flow cytometric-based approach that addresses the need for panels of cell-specific markers in the identification of immune cell populations, allowing both the accurate detection and quantitation of leukocyte subpopulations from a single, clinical kidney biopsy specimen. In ...
[摘要] 炎症性免疫细胞在急性肾损伤（AKI）和慢性肾病（CKD）的病例中发挥直接的病理作用。然而，人肾活组织检查中不同白细胞群的鉴定和表征已被用于检测它们的基于免疫组织化学（IHC）的技术的局限性所混淆。该方法不适用于明确定义离散免疫细胞群所必需的多种标志物的组合。我们开发了一种多参数，基于流式细胞仪的方法，解决了在鉴定免疫细胞群体时需要细胞特异性标记组，从而允许从单个临床肾活检标本中准确检测和定量白细胞亚群。在该方法中，将新鲜的人肾组织解离成单细胞悬浮液，然后进行抗体标记和基于流式细胞术的采集和分析。这种新技术为鉴定和计数人类肾脏疾病中的免疫细胞亚群提供了重大进步，并且是补充临床肾脏活组织检查的传统组织病理学检查的有力平台。
【背景】 肾脏疾病的发病率和患病率正在上升，给全世界的医疗保健社区带来了沉重的负担，并显着影响了患者的生活质量（世界肾脏日：慢性肾病，2015年）。肾脏损害可能由多种损伤引起，包括感染，缺血，毒素，高血压，遗传和代谢紊乱（Imig和Ryan，2013）。对功能性损伤的即时反应是急性肾损伤（AKI），这是一种临床综合征，其特征在于生理上通过肾功能的快速（数小时至数天）降低和组织形态学上通过炎性免疫细胞浸润到肾小管中，组织隔室邻接小管。肾脏（Basile ...
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