| Mouse CD8+ T Cell Migration in vitro and CXCR3 Internalization Assays
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Author:
Date:
2017-03-20
[Abstract] Chemokines are molecules that regulate the positioning of cells during homeostasis and inflammation. CXCL10 is an interferon-induced chemokine that attracts cells that express the chemokine receptor CXCR3 on their surface. CXCL10 expression is often induced upon inflammation and guides lymphocytes, such as T and NK cells, into the injured tissues. Notably, CXCL10 binding to CXCR3 induces receptor internalization and, therefore, low CXCR3 levels in cells positive for CXCR3 expression can be indicative of chemokine signaling.
Here, we describe an in vitro method to evaluate the ability of murine CD8+ T cells to migrate towards recombinant murine CXCL10; and a flow cytometry assay to measure CXCR3 expression levels at the surface of T cells, after exposure to ...
[摘要] 趋化因子是调节体内平衡和炎症期间细胞定位的分子。 CXCL10是干扰素诱导的趋化因子，其吸引在其表面上表达趋化因子受体CXCR3的细胞。 CXCL10表达通常在炎症诱导并引导淋巴细胞如T和NK细胞进入受损组织。值得注意的是，CXCL10与CXCR3结合诱导受体内化，因此CXCR3表达阳性细胞中的CXCR3水平降低可能是趋化因子信号传导的指示。
＆nbsp;这里，我们描述体外方法来评估鼠CD8 + T细胞向重组鼠CXCL10迁移的能力;以及暴露于不同剂量的趋化因子后在T细胞表面测量CXCR3表达水平的流式细胞术测定。
背景 趋化因子介导的T细胞运输是稳态和炎症期间的重要过程。活化的CD8 + T细胞表达趋化因子受体，例如CXCR3，允许它们向趋化因子CXCL9,10和11迁移，通常在损伤组织上上调。调节T细胞迁移的分子线索的评估对于了解其功能背后的生物学非常重要，但是在体内运行的复杂机制有时难以去卷积。在这里，我们提供有关体外方法的详细信息，以评估CD8 + T细胞上的趋化因子功能，重点是CXCL10介导的化学吸引和CXCR3内化。我们使用可以容易地在体外扩增和活化的抗原特异性转基因CD8 +细胞，因此提供足够数量的表型相同的淋巴细胞（例如， ...
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| Mouse Embryonic Fibroblast Cell Culture and Stimulation
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Author:
Date:
2016-07-05
[Abstract] Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity. Here, we describe procedures for mouse embryonic fibroblast cell isolation, primary culture, and stimulation. Specifically, we have optimized a step of serum starvation prior to stimulation. This step is necessary to ...
[摘要] 小鼠胚胎成纤维细胞（MEF）细胞的培养代表了测试基因功能的强大系统，因为它们容易获得，快速生长速率和大量实验的可能性。成纤维细胞是一组具有不同位置，不同外观和独特活动的间充质来源的异质驻留细胞。由于它们作为组织细胞的普遍分布，这些细胞准备响应由新激活的先天免疫细胞释放的因子，因此成为研究炎症和免疫的有用工具。在这里，我们描述了小鼠胚胎成纤维细胞分离，原代培养和刺激的程序。具体来说，我们已经优化了刺激前血清饥饿的步骤。这个步骤是必要的，以维持这些细胞在它们暴露于促炎刺激为最佳反应之前的静止状态。如在我们以前的研究中所示，这些小鼠成纤维细胞在通过常规Northern印迹技术容易检测的水平下不表达Tnf ， Csf2 或 Lai WS等人，2006）。
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