| Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices
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Author:
Date:
2018-04-05
[Abstract] We describe here a method to visualize concentration fields of cytokines around cytokine-secreting cells. The main challenge is that physiological cytokine concentrations can be very low, in the pico-molar range. Since it is currently impossible to measure such concentrations directly, we rely on cell’s response to the cytokines–the phosphorylation of a transcription factor–that can be visualized through antibody staining. Our devices aim at mimicking conditions in dense tissues, such as lymph nodes. A small number of secreting cells is deposited on a polylysine-coated glass and covered by multiple layers of cytokine-consuming. The cells are left to communicate for 1 h, after which the top layers are removed and the bottom layer of cells is antibody labeled for the response to cytokines. ...
[摘要] 我们在这里描述了一种可视化细胞因子分泌细胞周围细胞因子浓度场的方法。主要挑战是生理细胞因子浓度可能非常低，在微摩尔浓度范围内。由于目前不可能直接测量这样的浓度，我们依赖于细胞对细胞因子的反应 - 转录因子的磷酸化 - 可以通过抗体染色显现。我们的设备旨在模仿密集组织中的条件，如淋巴结。少数分泌细胞沉积在聚赖氨酸包被的玻璃上并被多层细胞因子消耗覆盖。将细胞连通1小时，之后去除顶层，并且细胞的底层被抗体标记为对细胞因子的应答。然后通过标准荧光显微镜观察细胞因子场的横截面。这篇手稿总结了我们的方法，以量化密集细胞体外细胞因子介导的细胞间通讯的程度。
【背景】哺乳动物的免疫系统已经发展到能够识别和限制潜在病原体的传播，同时使由免疫系统本身造成的附带组织损伤最小化。为了实现这一点，免疫细胞依赖细胞因子介质网络，这些细胞因子介质能够进行细胞间通讯并广播关于致病性侮辱的大小和性质的信息。大量不同细胞因子与其同源受体强烈结合，通常在纳摩尔或皮摩尔范围内具有特征性结合亲和力。通过细胞因子通讯产生免疫龛。例如，在骨髓和胸腺中，通过基质细胞分泌的白细胞介素-7（IL-7）分别支持增殖的B细胞和T细胞祖细胞的存活（Tokoyoda et al。， 2004; Alves等人，2009）。细胞因子生态位的大小控制成熟祖细胞的数量，从而保持血细胞区室平衡（Böyum，1968; ...
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| Murine in vitro Memory T Cell Differentiation
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Author:
Date:
2014-07-05
[Abstract] Upon pathogen encounter, naïve CD8+ T cells are primed and undergo massive clonal expansion. A fraction of effector CD8+ T cells remains during the contraction phase and differentiate into memory T cells critical for mounting robust recall responses in response to secondary infection. Low frequency of memory T cells in vivo is a major obstacle to investigate their functional aspects including migration capacity and genetic regulation. Here, we describe detailed protocol for memory T cell differentiation developed by von Andrian’s group to generate large number of CD44hiCD62Lhi antigen-specific memory T cells in vitro.
[摘要] 在病原体遭遇时，初始CD8 + T细胞被引发并经历大量克隆扩增。 效应CD8 + sup T细胞的一部分在收缩阶段期间保持并分化为响应于继发感染而对于安装鲁棒回忆反应至关重要的记忆T细胞。 体内低频记忆T细胞是研究其功能方面（包括迁移能力和遗传调节）的主要障碍。 在这里，我们描述由von Andrian的小组开发的记忆性T细胞分化的详细方案，以在体外产生大量的CD44 - 高 - 抗原特异性记忆T细胞 。
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| Murine in vivo CD8+ T Cell Killing Assay
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Author:
Date:
2014-07-05
[Abstract] Antigen-specific killing ability of effector CD8+ T cells is critical for protective immunity against infection. Here, we describe in vivo cytotoxic T cell assay to examine effector function of antigen-specific CD8+ T cells. Mice infected with Listeria monocytogenes (L. monocytogenes) expressing chicken ovalbumin as a model antigen mount ovalbumin-specific CD8+ T cell responses. Effector CD8+ T cell function in vivo is determined by mixed transfer of OVA peptide-pulsed target cells with control target cells into the previously immunized mice. Difference in CFSE expression levels clearly marks two distinct populations: Antigen-pulsed target cells-CFSElow vs. unpulsed target cells-CFSEhi. ...
[摘要] 效应CD8 + T细胞的抗原特异性杀伤能力对于感染的保护性免疫是关键的。 在这里，我们描述了在体内细胞毒性T细胞测定来检查抗原特异性CD8 + T细胞的效应子功能。 用表达鸡卵白蛋白作为模型抗原的单核细胞增生性李斯特菌（单核细胞增生李斯特氏菌）感染的小鼠装载卵清蛋白特异性CD8 + T细胞应答。 通过将OVA肽脉冲的靶细胞与对照靶细胞混合转移到先前免疫的小鼠中来测定体内效应物CD8 + T细胞功能。 CFSE表达水平的差异清楚地标记两种不同的群体：抗原脉冲的靶细胞-CFSE 低与未突发的靶细胞-FSE hi 。 抗原脉冲的靶细胞和对照靶细胞之间的频率用作抗原特异性杀伤的读数。
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