| In vitro Assessment of Immunological Synapse Formation by Flow Cytometry
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Author:
Date:
2016-03-20
[Abstract] In adaptive immune system, formation of immunological synapse between T cells and antigen presenting cells (dendritic cells, B cells, and macrophages) or target cells (tumor cells and viral-infected cells) is critical for the execution of T cell immune responses via cytokine secretion or direct killing activity. Here, we describe the practical methods that directly measure the number of conjugates as a result of immunological synapse formation between T cells and superantigen-loaded B cells or between cytotoxic T cells and antigen-loaded target cells by dual-color flow cytometry.
[摘要] 在适应性免疫系统中，T细胞和抗原呈递细胞（树突细胞，B细胞和巨噬细胞）或靶细胞（肿瘤细胞和病毒感染细胞）之间的免疫突触的形成对通过细胞因子执行T细胞免疫应答至关重要 分泌或直接杀死活动。 在这里，我们描述了通过双色流式细胞术直接测量T细胞和加载超抗原的B细胞之间或细胞毒性T细胞和抗原负载的靶细胞之间的免疫突触形成的结合物数量的实际方法。
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| In vivo Extravasation Assay
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Author:
Date:
2014-02-20
[Abstract] Tumor metastases develop when disseminated intravascular cancer cells acquire the ability to arrest by adhering to the capillary walls of distant organs, actively extravasate into their parenchyma, proliferate and establish secondary colonies. The extravasation assay described here is an in vivo technique aimed to analyze the ability of tumor cells to achieve early colonization of the lungs following tail vein injection in mice. Importantly, tumor cells need to be easily visible, therefore either they are fluorescent (e.g. expressing RFP or GFP) or they have to be pre-labelled with a fluorescent tracker prior to injection. Lungs are analyzed at different time points, experimentally determined by the researcher, depending on cell features and malignancy. Generally, an ...
[摘要] 当弥散性血管内癌细胞通过粘附到远端器官的毛细血管壁，主动外渗到其实质中，增殖和建立次级集落而获得阻滞的能力时，肿瘤转移发展。这里描述的外渗测定是体内技术，其目的在于分析肿瘤细胞在小鼠尾静脉注射后实现早期肺定植的能力。重要的是，肿瘤细胞需要容易可见，因此它们是荧光的（例如表达RFP或GFP），或者它们必须在注射之前用荧光跟踪器预标记。根据细胞特征和恶性肿瘤，在不同的时间点分析肺，由研究者实验确定。通常，需要早期时间点来检查针对注射的各种细胞的肺血管系统中的相等倒伏。在一个或多个稍后的时间点（6至48小时），定量分散在肺实质中的外渗细胞。使用我们的方案外渗在整个肺中直接评估离体考虑细胞荧光。然而，推荐内皮标记物的免疫荧光染色和肺切片的显微镜分析以评价肿瘤细胞（即在血管内或与其相关联的单个细胞或簇）内部，外部的定位和状态。由于外渗不仅受肿瘤细胞运动性而且受其存活能力的影响，用这种技术获得的结果应当与增殖和凋亡分析互补。
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