| 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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| Analysis of Tumor-infiltrating Lymphocytes Following CD45 Enrichment
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Author:
Date:
2014-08-20
[Abstract] Measuring antigen-specific T cell responses in the blood and lymphoid organs of vaccinated mice can give us a useful indication of the potency of a vaccine formulation. Unfortunately, systemic or even localized lymphoid T cell responses are not always predictive of the ability of a vaccine to induce tumor protection. Measuring the antigen-specific T cell response within the tumor infiltrating lymphocytes is a more accurate indicator or vaccine efficacy. However, multi-parameter flow cytometric analysis of T cells isolated from tumor tissue can be quite challenging due to the over-whelming number of tumor cells present in relation to the tumor infiltrating lymphocytes (TIL) and to problems associated to the large and adhesive nature of many tumor cell. Here we take advantage of a pre-flow ...
[摘要] 测量接种疫苗的小鼠的血液和淋巴器官中的抗原特异性T细胞应答可以给予我们疫苗制剂的效力的有用指示。不幸的是，全身性或甚至局部的淋巴T细胞应答并不总是预示疫苗诱导肿瘤保护的能力。测量肿瘤浸润淋巴细胞内的抗原特异性T细胞应答是更准确的指示或疫苗功效。然而，从肿瘤组织分离的T细胞的多参数流式细胞术分析可能是相当具有挑战性的，因为相对于肿瘤浸润性淋巴细胞（TIL）存在的肿瘤细胞的数量过多，以及与大的和粘附性质相关的问题的许多肿瘤细胞。在这里我们利用使用MACS磁性细胞分选系统从肿瘤组织中预流动分离CD45 +白细胞，产生更清洁的细胞制备物，进行流式细胞染色和分析。
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| Protocol for Preparation of Nuclear Protein from Mouse Lungs
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Author:
Date:
2014-06-05
[Abstract] This protocol describes how to extract nuclear protein from mouse lungs, including tissue preparation, stepwise lysis of cells and centrifugal isolation of nuclear protein fraction. This is an efficient method to get comparable nuclear protein extracts from lung tissues.
[摘要] 该协议描述如何从小鼠肺提取核蛋白，包括组织制备，逐步裂解细胞和核蛋白级分的离心分离。 这是从肺组织获得可比较的核蛋白提取物的有效方法。
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