Posch Wilfried, Lass-Flörl Cornelia, Wilflingseder Doris
Division of Hygiene and Medical Microbiology, Medical University of Innsbruck;
Division of Hygiene and Medical Microbiology, Medical University of Innsbruck.
J Vis Exp. 2016 Dec 24(118):54968. doi: 10.3791/54968.
Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to naïve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.
树突状细胞（DCs）通过其细胞表面表达的多种模式识别受体（PRRs）识别不同病原体的外来结构，如病毒、细菌和真菌，从而激活和调节免疫。DCs的主要功能是通过经由MHC I和MHC II分子将抗原呈递给幼稚T淋巴细胞，在淋巴结中诱导适应性免疫。因此，DCs在感染部位识别病原体后必须从外周迁移至淋巴结。对于体外实验或DC疫苗接种策略，通常使用单核细胞衍生的DCs。这些细胞在生理、形态和功能上与传统髓样树突状细胞相似。它们是通过用白细胞介素4（IL-4）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）刺激从健康供体分离的单核细胞而产生的。在此，我们展示了在通过密度梯度离心富集外周血单核细胞（PBMC）后，如何从抗凝血的人血中分离和刺激单核细胞。人单核细胞分化为未成熟DCs，孵育5天后即可在非临床环境中用于实验程序。
