Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
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DOI：
10.3791/54296
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发表时间：
2016-10-01
影响因子：
1.2
通讯作者：
Agudelo, Marisela
中科院分区：
文献类型：
--
作者：
Figueroa, Gloria;Parira, Tiyash;Agudelo, Marisela
树突状细胞是免疫系统的抗原呈递细胞，在淋巴细胞应答、宿主防御机制和炎症发病机制中起着至关重要的作用。由于树突状细胞的独特特性，其分离和研究在生物学研究中具有重要意义。虽然它们是免疫系统必不可少的关键介质，但dc在血液中非常罕见，约占血液单核细胞总数的0.1 - 1%。因此，替代的分离方法依赖于从外周血单核细胞（PBMCs）分离的单核细胞分化成树突状细胞。利用适当的分离技术，结合简单，负担得起，高纯度和高产量的细胞是必须考虑的。在目前的研究中，将比较两种不同的DCs生成方法。单核细胞通过粘附或磁分离过程负富集选择，然后用IL-4和GMCSF分化为DCs。利用活性染料、MTT法和成像流式细胞术分析CD11c/CD14表面标记物，评估单核细胞和MDDC的活力、增殖和表型。虽然磁分离法与黏附法相比，产生了更高百分比的单核细胞，具有更高的增殖能力，但研究结果表明，两种技术能够在培养7天后同时产生能够增殖和分化为有活力的CD11c+ mddc的单核细胞。两种方法的CD11c+ mddc产率均为70%。因此，我们的结果提供了有助于开发可靠的方法来分离和表征人类dc的见解。
Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GMCSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.