Isolation and Identification of Extravascular Immune Cells of the Heart
Isolation and Identification of Extravascular Immune Cells of the Heart
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DOI：
10.3791/58114
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发表时间：
2018-08-01
影响因子：
1.2
通讯作者：
Clemente-Casares, Xavier
中科院分区：
文献类型：
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作者：
Aronoff, Laura;Epelman, Slava;Clemente-Casares, Xavier
免疫系统是健康心脏的重要组成部分。心肌是不同免疫细胞亚群的丰富群体的家园，在稳态和不同形式的炎症期间具有功能区室化。直到最近，对心脏中免疫细胞的研究还需要使用显微镜或不发达的消化方案，这些方案在严重炎症期间提供了足够的灵敏度，但无法在稳态期间自信地识别小但关键的细胞群体。在这里，我们讨论了一个简单的方法相结合的酶（胶原酶，透明质酸酶和DNA酶）和机械消化的小鼠心脏之前，血管内给药的荧光标记抗体，以区分小，但不可避免的血管内细胞污染物。该方法产生分离的活细胞的悬浮液，其可以通过流式细胞术进行分析以进行鉴定、表型分析和定量，或者用荧光激活的细胞分选或磁珠分离进一步纯化以进行转录分析或体外研究。我们包括一个分步流式细胞术分析的例子，以区分心脏的关键巨噬细胞和树突状细胞群体。对于中等规模的实验（10个心脏），完成该程序需要2-3小时。
The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small - but key - populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2-3 h.