| ELISA on Virus-Infected Cells
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Author:
Date:
2014-05-20
[Abstract] The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells by endocytosis from the cell surface and fusion of the viral envelope with the membrane of late endosomes. The viral envelope glycoproteins undergo antigenic changes both upon virion endocytosis and upon fusion of the viral envelope with the endosomal membrane. These changes in virion antigenicity during virus entry were first described by immunofluorescence of infected cells. Although immunofluorescence provides valuable information on the subcellular distribution of the viral glycoproteins, the quantification of ...
[摘要] γ疱疹病毒鼠疱疹病毒4（MuHV-4）通过从细胞表面的内吞作用进入细胞并且病毒包膜与晚期内体膜的融合。病毒包膜糖蛋白在病毒颗粒内吞后和病毒包膜与内体膜融合时经历抗原性改变。病毒进入期间病毒体抗原性的这些变化首先通过感染细胞的免疫荧光来描述。尽管免疫荧光提供了关于病毒糖蛋白的亚细胞分布的有价值的信息，但是大量细胞中免疫荧光信号的定量不仅依赖于相对昂贵的显微镜设备，而且相对耗时。为了以可靠，以及时间和成本有效的方式定量进入NMuMG上皮细胞的MuHV-4病毒粒子的抗原性，我们开发了具有感染细胞作为固相的ELISA。在该测定中，细胞在96孔组织培养板上生长，在4℃暴露于病毒粒子，然后在37℃孵育，允许病毒粒子内吞。细胞在病毒体结合后直接在4℃固定，或在37℃孵育后固定。在随后的透化作用之后，将细胞与对病毒包膜糖蛋白特异性的单克隆抗体温育，然后用碱性磷酸酶偶联的二抗检测。在用磷酸硝基苯酯底物孵育细胞时，在常规ELISA酶标仪上测量吸光度。讨论了不同的数据解释方式。
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| Endosomal pH Measurement in Bone Marrow Derived Dendritic Cells
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Author:
Date:
2013-07-20
[Abstract] Endosomes embraces different set of compartments such as early endosomes, intermediate endosomes and late endosomes or lysosomes. They become acidic as they mature. This acidification is generated by the vacuolar membrane proton pump V-ATPase that is recruited in late endosomes. This protocol described the measurement of endosomal pH using dextran molecules labelled with pH sensitive and insensitive dyes.
[摘要] 内体包含不同组的区室，例如早期内体，中间内体和晚期内体或溶酶体。 它们成熟时变成酸性。 这种酸化由在后期内体中招募的液泡膜质子泵V-ATP酶产生。 该协议描述了使用用pH敏感和不敏感染料标记的葡聚糖分子测量内体pH。
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| Isolation of Phagosomes from Dendritic Cells by Using Magnetic Beads
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Author:
Date:
2013-07-20
[Abstract] Phagosomes are intracellular organelles in dendritic cells in which pathogens such as viruses, bacteria and parasites are internalised to be proteolysed and killed. Phagosomes are formed by fusion with the plasma membrane, some area of the endoplasmic reticulum as well as the lysosome. This protocol described the purification of phagosomal compartments at different stage of their maturation using magnetic beads.
[摘要] 吞噬体是树突细胞中的细胞内细胞器，其中病原体例如病毒，细菌和寄生虫被内化以被蛋白水解和杀死。 吞噬体通过与质膜，内质网的一些区域以及溶酶体融合形成。 该协议描述了使用磁珠在其成熟的不同阶段的吞噬体隔室的纯化。
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