Genetic Modification of Primary Human Myeloid Cells to Study Cell Migration, Activation, and Organelle Dynamics.
Genetic Modification of Primary Human Myeloid Cells to Study Cell Migration, Activation, and Organelle Dynamics.
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DOI：
10.1002/cpz1.514
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发表时间：
2022-08
期刊：
影响因子：
--
通讯作者：
Johnson, Jarrod S
中科院分区：
文献类型：
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作者：
Greiner, Daniel;Scott, Tiana M;Olson, Gregory S;Aderem, Alan;Roh-Johnson, Minna;Johnson, Jarrod S
髓样树突状细胞（DC）和巨噬细胞是在免疫系统中具有关键作用的单核吞噬细胞。作为抗原呈递细胞，它们将微生物的先天检测与编程适应性免疫反应联系起来。髓样DC和巨噬细胞也在发育中发挥关键作用，促进组织稳态，并响应损伤和炎症直接修复。由于细胞迁移和细胞器动力学与这些过程密切相关，因此有必要开发跟踪骨髓细胞行为和功能的工具。在这里，我们建立在以前建立的协议，从外周血中分离原代人髓系细胞，并报告了一种优化的方法，其基因修饰慢病毒载体，以研究与细胞迁移，激活和细胞器动力学的过程。具体而言，我们提供了一种用于将遗传编码的荧光标记物递送到原代单核细胞衍生的树突状细胞（MDDC）和单核细胞衍生的巨噬细胞（MDM）中以标记线粒体、过氧化物酶体和全细胞的方案。我们描述了使用磁珠阳性选择从外周血中分离初级CD14+单核细胞，或者基于塑料粘附分离。分离的CD14+细胞可以用慢病毒载体转导，随后在细胞因子存在下培养以衍生MDDC或MDM。该方案高度适用于与载体共转导以敲低或过表达感兴趣的基因。这些工具使得能够通过流式细胞术、荧光显微镜和其他下游测定来进行遗传修饰的骨髓细胞的机制研究。
Myeloid dendritic cells (DCs) and macrophages are mononuclear phagocytes with key roles in the immune system. As antigen presenting cells, they link innate detection of microbes with programming adaptive immune responses. Myeloid DCs and macrophages also play critical roles in development, promote tissue homeostasis, and direct repair in response to injury and inflammation. As cellular migration and organelle dynamics are intimately connected with these processes, it is necessary to develop tools to track myeloid cell behavior and function. Here, we build on previously established protocols to isolate primary human myeloid cells from peripheral blood and report an optimized method for their genetic modification with lentiviral vectors to study processes related to cell migration, activation, and organelle dynamics. Specifically, we provide a protocol for delivering genetically-encoded fluorescent markers into primary monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs) to label mitochondria, peroxisomes, and whole cells. We describe the isolation of primary CD14+ monocytes from peripheral blood using positive selection with magnetic beads, and alternatively, isolation based on plastic adherence. Isolated CD14+ cells can be transduced with lentiviral vectors and subsequently cultured in the presence of cytokines to derive MDDCs or MDMs. This protocol is highly adaptable for co-transduction with vectors to knockdown or overexpress genes of interest. These tools enable mechanistic studies of genetically modified myeloid cells through flow cytometry, fluorescence microscopy, and other downstream assays.