| Murine Monocyte and Macrophage Culture
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Author:
Date:
2021-03-20
[Abstract] Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past to differentiate myeloid cell types from bone marrow cells, enabling functional investigation and furthering our understanding about their contribution to mammalian physiology. In this protocol, we describe a simple and rapid method to isolate monocytes from murine bone marrow, culture them for up to 5 days and lastly, differentiate them into bone marrow derived macrophages (Figure 1).
Graphic abstract: ...
[摘要] [摘要]骨髓中的骨髓祖细胞产生单核细胞，巨噬细胞，粒细胞和大多数树突状细胞。即使这些先天免疫细胞是同一谱系的一部分，每种细胞类型在组织发育，宿主防御和适应性免疫的产生中也发挥着特定而关键的作用。过去已经开发出区分骨髓细胞和骨髓细胞的协议，以进行功能研究并加深我们对它们对哺乳动物生理学贡献的理解。在该协议中，我们描述了一种简单快速的方法，可从鼠骨髓中分离单核细胞，将其培养长达5天，最后，将它们分化为源自骨髓的巨噬细胞（图1）。
图形摘要：
图1.实验概述，描绘了鼠单核细胞和巨噬细胞培养的步骤
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| Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting
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Author:
Date:
2016-11-20
[Abstract] In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 x 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes.
[摘要] 在这个协议，我们描述从初始小鼠的肺分离高纯度原发性肺泡上皮细胞类型（ATII）细胞的方法。该方法结合对多种谱系标志物的阴性选择以及对于Ep上皮细胞标记物EpCAM的阳性选择。该方法每小鼠肺产生2-3×10 6个ATII细胞。细胞制品是高度纯的和可行的，并且可以用于基因组或蛋白质组分析或培养离体以了解他们在各种生物过程中的作用。
[背景] 肺的内表面由上皮细胞排列，上皮细胞的类型在形态上和功能上随着肺内的位置而变化。 ATII细胞是两种类型的上皮细胞中的一种，其排列在肺泡壁上并且已经被描述为在表面活性剂合成和分泌中起关键作用。它们也是肺内第一道防线的一部分，并且涉及在肺部感染或过敏期间引发和调节免疫应答。它们还被认为在远端肺中充当具有增殖能力和损伤后修复上皮的能力的祖细胞。 ATII分离的可用方法不产生超过80-85％纯度的细胞制品，使得它们不适合于mRNA和蛋白质表达的可靠分析。本文所述的方法是对现有方法的改进，并产生具有最高纯度的小鼠原代ATII细胞制备物，因此可以可靠地用于表达分析。对于该方法的进一步讨论，我们将读者指向该协议起源的原始出版物（Sinha等人，2016）。
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| Vaccine-induced Cytokine Production Detected by Luminex Multiplex Analysis
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Author:
Date:
2014-08-20
[Abstract] Different vaccine and adjuvant combinations are known to rapidly induce antigen presenting cell (APC) maturation and pro-inflammatory cytokine and production, which in turn play an important role in the priming of antigen-specific T cells. Measuring cytokine production systemically in the serum fails to detect localized responses in the lymph nodes draining a subcutaneous immunization site. On the other hand, stimulating APC with vaccine formulations in vitro lacks the complexity of the lymph node microenvironment and the presence of other in vivo factors. Here we analyse cytokine production directly in vaccine draining lymph nodes (dLN) extracted early after in vivo vaccination. To do this we perform cytokine multiplex analysis of supernatants from whole dLN ...
[摘要] 已知不同的疫苗和佐剂组合快速诱导抗原呈递细胞（APC）成熟和促炎细胞因子和产生，其反过来在抗原特异性T细胞的引发中起重要作用。 在血清中全身测量细胞因子产生不能检测排出皮下免疫位点的淋巴结中的局部反应。 另一方面，用疫苗制剂在体外刺激APC缺少淋巴结微环境的复杂性和其他体内因子的存在。 在这里我们分析细胞因子生产直接在疫苗引流淋巴结（dLN）提前早期在体内接种。 为此，我们在短暂的离体孵育后对来自整个dLN细胞悬浮液的上清液进行细胞因子多重分析。
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