| Spectrophotometric Determination of Glutamine Synthetase Activity in Cultured Cells
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Author:
Date:
2016-10-05
[Abstract] Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and Wilmore, 1990). Glutamine is involved in many biological processes including serving as the nitrogen donor for biosynthesis, as an exchanger for the import of essential amino acids, as a means to detoxifying intracellular ammonia and glutamate, and as a bioenergetics nutrient to fuel the tricarboxylic acid (TCA) cycle (Bott et al., 2015). The method for the assay of GS enzymatic activity relies on its γ-glutamyl transferase reaction by ...
[摘要] 谷氨酰胺合成酶（GS），其催化谷氨酸和氨转化成谷氨酰胺，广泛分布在动物组织和细胞培养系中。该酶的重要性通过谷氨酰胺，GS-催化的从头合成反应的产物，是血液中最丰富的游离氨基酸的事实提示（Smith和Wilmore，1990）。谷氨酰胺参与许多生物过程，包括作为生物合成的氮供体，作为输入必需氨基酸的交换剂，作为解毒细胞内氨和谷氨酸的手段，以及作为生物能量营养物来给三羧酸（TCA）周期（Bott等人，2015）。用于测定GS酶活性的方法依赖于其γ-谷氨酰转移酶反应，通过测量由谷氨酰胺和羟胺合成的γ-谷氨酰羟肟酸酯，以及反应产物与反应物的色谱分离（Deuel等人 。，1978）。 GS谷氨酰转移酶反应的概述可以在图1中找到。通过分光光度测定法在560nm的特定波长下使用酶标仪测量GS活性。该方法简单，并且具有与应用放射性标记的底物的那些方法相当的灵敏度。该修改的方法已经应用于在包括人乳腺上皮MCF10A细胞和鼠前B FL5.12细胞的培养细胞系中测定/测定GS活性，并且可以用于测量其他细胞系中的GS活性。 >
图1 。GS glutamyl ...
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| Isolation and in vivo Transfer of Antigen Presenting Cells
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Author:
Date:
2014-10-05
[Abstract] Transfer of antigen presenting cells in vivo is a method used by immunologists to examine the potency of antigen presentation by a selected population of cells. This method is most commonly used to analyze presentation of protein antigens to MHC class I or II restricted T cells, but it can also be used for studies of nonconventional antigens such as CD1-presented lipids. In a recent study focusing on CD1d-restricted glycolipid antigen presentation to Natural Killer T cells, we compared antigen presenting properties of splenic B cells, CD8αPos dendritc cells (DCs) and CD8αNeg DCs (Arora et al., 2014). This protocol describes the detailed method used for isolation of these cell populations, and their transfer into recipient mice to analyze their ...
[摘要] 体内转移抗原呈递细胞是免疫学家用来检查所选择的细胞群体的抗原呈递效力的方法。该方法最常用于分析蛋白质抗原对MHC I类或II类限制性T细胞的呈递，但其也可用于非常规抗原如CD1呈递脂质的研究。在最近关于CD1d限制性糖脂抗原呈递到天然杀伤T细胞的研究中，我们比较脾B细胞，CD8αpos 树突细胞（DC）和CD8α Neg sup> DC（Arora et al。，2014）。该方案描述了用于分离这些细胞群体和将其转移到受体小鼠中以分析其抗原呈递性质的详细方法。作为总单核细胞的百分比，平均脾脏含有约1-3％骨髓树突状细胞（DC）。在绝对数量上，这转换为大约0.6-1.8×10 6个DC。为了增强脾中DC的数目，向小鼠皮下注射来自经过工程改造以表达fms相关的酪氨酸激酶3配体（Flt3L）的培养的黑素瘤细胞系（B16.Flt3L）的细胞（Mach等人。，2000）。该蛋白是与集落刺激因子-1同源的生长因子，并在造血干细胞的分化中起关键作用。将这种蛋白质作为纯化的蛋白质施用到小鼠中导致多个器官中的CD8αpos pos和CD8α阳性DC亚群的扩增。在已经植入过表达这种蛋白质的肿瘤细胞的小鼠中也观察到类似的扩增（Mach等人，2000）。根据我们的经验，来自具有可触知的B16.Flt3L肿瘤的小鼠的脾脏中高达60％的总单核细胞可以是CD11c阳性树突细胞，从而得到高达5×10 ...
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| Adhesion of Moraxella catarrhalis to Respiratory Tract Epithelial Cells
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Author:
Date:
2013-11-05
[Abstract] Moraxella catarrhalis is a human-restricted pathogen that is responsible for respiratory tract infections such as childhood otitis media (OM) and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Successful colonization and infection by M. catarrhalis depends on its ability to attach to the respiratory tract mucosal epithelium. This protocol describes a method to measure adherence of M. catarrhalis to epithelial cell lines in vitro.
[摘要] 卡他莫拉菌（Moraxella catarrhalis）是一种人类限制性病原体，其负责呼吸道感染，例如成年儿童中耳炎（OM）和慢性阻塞性肺病（COPD）的加重。 成功殖民和感染由M. 卡他性取决于其附着到呼吸道粘膜上皮的能力。 该协议描述了测量M的依从性的方法。 卡他莫利斯到上皮细胞系。
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