Application of six-color flow cytometry for the assessment of dendritic cell responses in whole blood assays
Application of six-color flow cytometry for the assessment of dendritic cell responses in whole blood assays
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DOI：
10.1016/j.jim.2008.09.009
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发表时间：
2008-12-31
影响因子：
2.2
通讯作者：
Villa, Maria Luisa
中科院分区：
文献类型：
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作者：
Della Bella, Silvia;Giannelli, Stefania;Villa, Maria Luisa
外周血树突状细胞(PBDC)的分析在广泛的病理学中越来越具有临床意义，其中研究DC亚群对特定刺激的充分反应的能力可能有助于理解潜在的免疫病理机制。与其他需要DC分离和培养的方法相比，直接在全血(WB)样本中评估PBDC反应具有许多优势，但它在多参数分析中受到限制，目前基于三色或四色分析。因此，在这项研究中，我们开发了一种6色分析方法，专门用于分析PBDC对WB刺激的反应。我们将WB样本与具有明确分布的Toll样受体(Toll-like Receptor，TLR)的配体孵育在髓系树突状细胞(MDCs)或浆细胞样树突状细胞(PDCs)上，并分析DC在激活/成熟标志物上调以及产生广泛调节细胞因子方面的反应。用四种颜色检测CD80、CD86、CD40、CD83的表达或细胞内IL-12、肿瘤坏死因子(TNF)-α、干扰素(IFN)-α、IL-6、IL-10、IL-4的积聚。利用这种方法，我们可以在同一流式细胞仪上直接比较MDCs和PDCs对TLR刺激的反应，并研究不同的激活标志或调节细胞因子的相互共表达。我们认为，本文提出的6色WB法可能是研究DC复杂生物学的一种新工具。(C)2008爱思唯尔B.V.保留所有权利。
Analysis of peripheral blood dendritic cells (PBDCs) is increasingly reaching clinical relevance in a wide range of pathologies, in which investigating the capacity of DC subsets to respond adequately to specific stimuli may aid the comprehension of underlying immunopathologic mechanisms. The evaluation of PBDC responses directly challenged in whole blood (WB) samples offers many advantages over other methods that require DC isolation and culture, but it is limited in multiparametric analysis, currently based on 3- or 4-color assays. Therefore, in this study we developed a 6-color assay dedicated to the analysis of PBDC responses upon WB stimulation. We incubated WB samples with ligands to toll-like receptors (TLRs) with a clear-cut distribution on myeloid DCs (mDCs) or plasmacytoid (pDCs) and analyzed DC responses in terms of upregulation of activation/maturation markers, as well as production of a wide range of regulatory cytokines. Four colors were used to gate on mDCs and pDCs that were identified as lineage-/HLA-DR+ICD11c+ and lineage-/HLA-DR+/CD123+, respectively, and two further colors were used to analyze either the surface expression of CD80, CD86, CD40 or CD83, or the intracellular accumulation of IL-12, tumor-necrosis factor (TNF)-alpha, interferon (IFN)-alpha, IL-6, IL-10 or IL-4. With this method, we could directly compare in the same flow cytometric tube the responses of mDCs and pDCs to TLR stimulation, and investigate the reciprocal coexpression of distinct activation markers or regulatory cytokines. We suggest that the 6-color WB assay presented here may represent a novel tool for investigating the complex biology of DCs. (C) 2008 Elsevier B.V. All rights reserved.