| Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs
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Author:
Date:
2016-11-05
[Abstract] This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTrackerTM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than ...
[摘要] 该方案是基于流式细胞术的方法，以测量骨髓来源的树突细胞（BMDCs）在体外的坏死性靶细胞的吞噬效率（Aaes等人 2016）。该方法是先前发表的论文中使用的方案的稍微修改和更新的版本（Krysko等人，2006; Brouckaert等人，2004）。简言之，在细胞被诱导经历细胞死亡之前，用CellTracker TM TM染料标记靶细胞。在用BMDCs共培养2小时后，用树突细胞标记物和死细胞标记物对细胞进行免疫染色，并使用流式细胞仪定量吞噬效率。该方案可以容易地用于经历除坏死作用以外的细胞死亡模式的靶细胞。
[背景] 研究BMDCs吞噬细胞摄取的坏死细胞是检测免疫原性细胞死亡模型的初步步骤（Obeid等人，2007）。有效摄取将允许吞噬细胞将抗原交叉呈递到白细胞，从而产生针对死的靶细胞的免疫反应。在该协议中，我们使用CellTracker TM sup TM染料。这种类型的染料在某些浓度下可能是有毒的，其可以根据使用的细胞类型而变化。因此，我们建议用户首先为使用的靶细胞找到最佳的染料浓度。最佳地，CellTracker TM 染料本身不应该诱导任何细胞死亡，而是应当标记靶细胞，使得它们容易与CD11c阳性BMDC分离。
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| 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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