| 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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| Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay
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Author:
Date:
2017-11-20
[Abstract] Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes. Golgi membranes were therefore isolated from GalT-CFP expressing cells, while vesicles were prepared from GalT-YFP expressing ones. Incubating CFP-labelled Golgi with YFP-labelled vesicles in the presence of cytosol and an energy regeneration mixture at 37 °C produced a ...
[摘要] 高尔基体内的逆行囊泡运输被用来运送和分类高尔基酶到适当的池内位置。建立了一个检测方法来研究无细胞系统中囊泡靶向的分子细节。生成了稳定的细胞系，其中反式 - 高尔基酶半乳糖基转移酶（GalT）用CFP或YFP标记。考虑到GalT被循环到稳定状态的小池中，含有GalT的囊泡将靶向含有GalT的池内膜。因此从表达GalT-CFP的细胞分离高尔基体膜，而从GalT-YFP表达细胞制备囊泡。在胞质溶胶和能量再生混合物的存在下，在37℃孵育CFP标记的高尔基体和YFP标记的囊泡，与在冰上孵育相比，在混合物的荧光成像后CFP-YFP共定位显着增加。该测定被验证需要能量，蛋白质和生理学重要的运输组分，如Rab GTP酶和保守寡聚体高尔基体系复合物。该测定法可用于调查影响高尔基体运输机器的生理和病理变化，特别是囊泡束缚。
【背景】细胞内囊泡靶向的分子机制对于解释糖基化稳态，神经递质释放，信号受体的调节和营养摄取等方面的解释是重要的（Ungar和Hughson，2003; ...
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| Plasmodium Sporozoite Motility on Flat Substrates
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Author:
Date:
2017-07-20
[Abstract] Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.
[摘要] 疟原虫子孢子是由按蚊按蚊传播的疟疾寄生虫的传染性，高度活跃的形式。 可以在唾液腺分泌唾液腺和体外分离子孢子之后评估子孢子运动性。
【背景】疟原虫的病原体疟原虫的子孢子通过感染性蚊子的叮咬传播到其脊椎动物宿主的皮肤中。 子孢子运动是寄生虫传播和成功感染脊椎动物宿主的关键前提。 动力构成第一个可以被抑制的寄生虫机制，因此对于干预策略是有意义的。 影响滑动运动性或运动性调节化合物的遗传修饰可以使用体外实验来容易地进行研究。
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