通过成像流式细胞仪人单核细胞来源的树突状细胞的特征：两个单核细胞分离方法的比较
Summary
This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.
Abstract
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 GM-CSF. 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.
Introduction
树突细胞（DC）是先天和适应性免疫系统的基本介质。它们的功能来诱导初次免疫应答，促进免疫记忆的发展。这些细胞对于抗原捕获，迁移和T细胞的刺激主要负责，因此，被称为1的DC .Manipulation可以在各种不同的研究领域，并且在临床上被用于治疗不同的专业抗原呈递细胞（APC）炎性疾病如HIV 6,7，癌症8，自身免疫性疾病9，和过敏性反应10。区议会也被用于药物滥用研究，以解决未知的机制和途径，如与酒精依赖11-14，药物依赖13,15，和HIV感染和滥用药物16-19的组合有关的。这些正在进行的研究和未来研究研究我ñ免疫学领域作出的体外代DCS的研究非常重要。然而，存在与从人血液中分离的DC，因为它们只构成0.1相关联的若干困难-总血单核细胞20的1％。
迄今为止，一些对的DC 在体外的产生的行之有效的方法是由单核细胞21,22的塑料或玻璃粘附，密度梯度离心23，特异性标记基于分离的诸如磁激活细胞分选22，荧光激活细胞分选24，用葡聚糖包被的磁性纳米颗粒25，并用完全自动化的负小区选择26高度纯化的单核细胞的快速分离CD14 +单核细胞的阳性筛选。但是，选择的最佳方法仍存在争议。因此，为了改善直流生成技术，几种方法已经被开发，其中这些细胞的纯度，可以大大增加从外周血单核细胞（PBMC）27分离纯化的CD34 +祖细胞和单核细胞的分化。作为现有所提到的，用于产生单核细胞来源的树突细胞（MDDCs）一种广泛使用的和流行的方法是探索的单核细胞粘附到玻璃或塑料（附着法）21,22,27的能力。的粘附方法是，不需要使用复杂的设备迅速和直接的方法。然而，这种方法的一些缺点包括淋巴细胞污染，低弹性，和单核细胞的瞬态操作28。到的粘附方法的另一种方法是从总的PBMC的单核细胞的磁隔离，特别是与使用人单核细胞富集的试剂盒，其被设计为通过阴性选择26来隔离的PBMC的单核细胞的。在此过程中，不想要的细胞被定位为与四聚体去除抗体复合物和葡聚糖 – 包被的磁性粒子。这种分离方法的优点是，不希望的标记的细胞使用磁体而靶细胞可以自由地倾出进新管中，而不需要的列隔开。迄今为止，与可标记独特的细胞群体特异性的单克隆抗体的情况中，磁性分离技术已成为不只是一个附加的方法，但对于罕见细胞在免疫学领域的隔离是必要的。例如，技术，诸如磁性细胞与市售的顺磁磁珠纳米颗粒分拣提供了便利的研究和临床应用22,29的新方法的发展。此外，最近的调查研究DC一代单核细胞，从坚持和MACS技术方法相比采用分离的单核细胞22,30 MACS都表现出较高的DC纯度和可行性。
目前的研究礼物使用商业人单核细胞富集试剂盒1）通过粘附的单核细胞的分离和2）单核细胞分离通过阴性选择：两种方法人DCs的一代从单核细胞从PBMC中分离之间的比较。本研究提供的证据表明，当与由粘附方法分离的单核细胞相比，阴性选择磁选步骤以分离单核细胞产生具有在单核细胞存活率没有显著差异的单核细胞的产率最高。反过来，七天后，通过磁分离中分离出的单核细胞分化成与显著更高的增殖能力和表达双阳性（的CD11c + / CD14 +）表型的细胞的较高量MDDCs而不影响MDDC生存能力。总体而言，目前的研究从上面引用的先前研究不同，因为它表明这两种技术以同时产生能够增殖和分化成的CD11c的单核细胞的能力+MDDCs（> 70％）后，在培养7天不影响他们的生存能力。此外，目前的做法提供了流式细胞仪成像流量不同的CD11c / CD14 MDDCs人口的第一次鉴定。
综上所述，由于区议会扮演关于免疫学领域研究的焦点角色，不同的参数必须考虑到他们是如何得出的，并用什么方法将它们在体外分离培养时加以考虑。因此，本研究的目的是提供对单核隔离两种不同的方法，以及这些方法的差异影响单核细胞活力和最终产量影响树突状细胞活力，增殖和表型的见解。这些发现将大大有助于免疫学的领域，并且将提供的DC隔离，纯化和表征的详细协议。
Protocol
Representative Results
Discussion
根据分离和产生从人血MDDCs的已知困难，本研究的目的是提供用于MDDCs的产生两个成熟的方法的全面的比较。相比第一种方法是用于通过利用单核细胞粘附到玻璃或塑料（附着法）21,22,27的能力产生MDDCs行之有效的传统方法。的粘附方法是快速和成本有效的，并且不要求使用复杂的设备。然而，这种方法的一些缺点包括淋巴细胞污染，低弹性，单核细胞瞬时操纵22,30,31。相比，第二?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This research is supported by the National Institute on Alcohol Abuse and Alcoholism, award K99/R00 AA021264. Additional lab support as part of startup package has been received from the Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, and FIU- Office of Research and Economic Development. Gianna Casteleiro was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Materials
| Ficoll-Paque | GE Healthcare | 17-5442-03 | Must be used at room temperature |
| Phosphate-buffered saline (PBS) | Life Technologies | 10010-023 | |
| ACK Lysing buffer | Quality Biological | 118-156-101 | |
| RPMI 1640 medium | Life Technologies | 22400-089 | |
| Antibiotic-Antimycotic (100X) | Life Technologies | 15240-062 | |
| Fetal Bovine Serum (FBS) | Life Technologies | 16000-044 | |
| RoboSep buffer | StemCell | 20104 | |
| EasySep Human monocyte enrichment kit | StemCell | 19059 | |
| TC20 Automated cell counter | Bio-Rad | 145-0101 | |
| FITC-Dextran | Sigma Aldrich | FD4-100MG | |
| Trypan blue stain (0.4%) | Life Technologies | 15250-061 | |
| Synergy 2 multi-mode reader | Biotek | 7131000 | |
| XTT Sodium salt bioreagent (XTT) | Sigma Aldrich | X4626-100MG | |
| Dimethyl sulfoxide bioreagent (DMS) | Sigma Aldrich | D8418-500ML | |
| Thiazolyl blue tetrazolium bromide (MTT) | Sigma Aldrich | m5655-500MG | |
| Sodium Dodecyl Sulfate (SDS) | Bio-Rad | 161-0302 | |
| Phenazine Methosulfate (DMSO) | Sigma Aldrich | P9626-1G | |
| Inactivated (HI) Human Serum | Chemicon | S1-100ML | |
| Accuri C6 Flow Cytometer | BD Accuri | 653119 | |
| FlowSight Amnis Flow Cytometer | EMD Millipore | 100300 |
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