脂肪组织免疫细胞的分离
Summary
脂肪组织（AT）是一个强烈的免疫细胞的激活和相互作用的部位。几乎所有的免疫系统的细胞是存在于AT和它们的比率是改变了肥胖。适当的隔离，定量，定性的AT免疫细胞群，是了解自己的角色在immunometabolic疾病的关键。
Abstract
在脂肪组织中的巨噬细胞浸润增加（AT）的肥胖啮齿类动物和人类的发现感兴趣的免疫细胞贡献的加剧，导致局部和全身的胰岛素抵抗。瘦肉和肥胖不同的免疫细胞群的分离和量化是现在普遍使用的技术，还必须格外小心，应采取使血管基质细胞的分离和流式细胞仪分析获得的数据是可靠的，可解释的在immunometabolism实验室。在这段视频中，我们展示了如何切末，消化，并隔离富含免疫细胞间质血管分数。随后，我们将展示如何抗体标记巨噬细胞和T淋巴细胞，以及如何正确的门对他们在流式细胞仪实验。代表流式细胞仪地块从低脂肪喂养的瘦和高脂肪喂养的肥胖小鼠。在此分析中的一个关键因素是使用的抗体，该抗体荧光渠道在巨噬细胞是自然自发荧光，以及使用适当的补偿控制。
Introduction
从历史上看，脂肪组织（AT）一直被看作是惰性的脂质储存器官，膨胀和收缩响应能量平衡。我们现在明白了，在代表一个动态的内分泌器官，积极分泌一些激素，它直接影响摄食行为和全身葡萄糖稳态。此外，在过去十年中一直为众多的人口居住在血管基质部分（SVF），以及他们的贡献动态平衡免疫细胞的不断升值。
其次通过差速离心分离脂肪细胞和SVF使用胶原酶消化能力罗德贝尔首次描述了在1964年1。 Ⅱ型胶原酶是最常用的为脂肪细胞的和SVF分离的脂肪细胞的胰岛素受体1的维护。在早期，主要采用酶分馏AT学习体重减轻，脂肪CYTE代谢和隔离脂肪前体细胞。最近，这种技术，结合流式细胞仪和广泛的可用性市售荧光标记的抗体的数量不断增加，促进免疫细胞的表征AT。
虽然存在前面所述的免疫细胞在发炎AT 2，Weisberg的等人的开创性论文。徐等人 。发表于2003年首次记录积累的巨噬细胞（自动取款机）肥胖，分泌炎性细胞因子，并与AT特异性和全身胰岛素抵抗3,4。这些观测服务为基础，最近创造了一个新的研究领域，“immunometabolism，”5，并已跟进研究，涉及各种免疫细胞的人口，包括树突状细胞，肥大细胞，T细胞8 –10，B细胞，NKT细胞，嗜酸性粒细胞13，与肥胖相关的胰岛素抵抗的发展中的嗜中性粒细胞14,15。
这篇文章的目标是提供胶原酶消化技术，用于分离细胞的AT SVF和表征自动取款机和AT＆T的细胞通过流式细胞仪的详细说明。该协议已优化的鼠标，但是，观众可能会受益于阅读的优秀文章，优化这一技术为人类提供丰富的细节在16。这篇文章的目标受众包括有限的工作经验，用鼠标进行流式细胞仪的调查。平衡的时间和资源的细胞产率以及活性与几个实际的考虑，以及最佳的流式细胞仪控制AT的免疫细胞群的特征。除了我们的通讯协议，读者请参照最近的朱庇特的文章Basu 等的精彩讨论一些流式细胞仪技术方面的，包括适当的控制和补偿17。
Protocol
Representative Results
Discussion
流式细胞仪的广泛使用，表征免疫细胞的AT导致肥胖的代谢结果，在免疫系统中的作用越来越大的兴趣。虽然确切的协议会有所不同，根据自己的经验和现有设备的实验室之间的关键步骤包括：胶原酶消化，差速离心和细胞表面抗原的标记。本文的目标是提供一个详细的协议和实用指南隔离AT SVF有限的工作经验，与AT和/或执行流式细胞仪调查。
任何实验技术，有无数的变化，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
JSO支持由美国国立卫生研究院露丝，属Kirschstein NRSA（F32 DK091040），AK是来自美国糖尿病协会（7-10-MI-05）的博士后奖学金支持，AHH支持由美国心脏协会成立研究者奖（12EIA8270000）。流式细胞仪检测实验进行了流式细胞仪在VMC共享资源。 VMC流式细胞仪共享资源支持范德比尔特消化疾病研究中心（DK058404）。
Materials
| Name | Company | Catalog # | |
| DPBS (no Ca or Mg) 10 x 500 ml | Life Technologies | 14190-250 | |
| DAPI | Life Technologies | D3571 | |
| BSA | Sigma | A2153-100G | |
| collagenase | Sigma | C6885-5G | |
| propidium iodide solution | Sigma | P4864-10 ml | |
| stable stack 20 microliter | Rainin | SS-L10 | |
| 20 microliter filter tips | Rainin | SRL10F | |
| stable stack 250 microliter tips | Rainin | SS-L250 | |
| 1000 microliter tips | Rainin | GPS-L1000 | |
| 1000 microliter filter tips | Rainin | GP-L1000F | |
| 250 microliter Filter Tips | Rainin | SR-L200F | |
| FC Block | BD Biosciences | 553142 | |
| fisher 100mn strainers | Fisherbrand | 22-363-549 | |
| medium weigh dish | Fisherbrand | 02-202B | |
| aluminum foil | Fisherbrand | 1213100 | |
| mincing scissors | Fisherbrand | 089531B | |
| Vortex | Fisherbrand | 2215365 | |
| 50 ml conical tubes | BD Falcon | 14-959-49A | |
| filter top FACS tubes | BD Falcon | 352235 | |
| 10 ml pipette case 200 | BD Falcon | 1367520 | |
| round bottom tubes | BD Falcon | 352058 | |
| 5 ml syringe | BD Falcon | 309646 | |
| V Bottom Plates | Costar | 07-200-107 | |
| transfer bulb pipette | Thermo Scientific | 13-711-22 | |
| Shaker | Thermo Scientific | 11 676 071 | |
| Adhesive mat | Thermo Scientific | 1368750 | |
| Cell Culture Centrifuge | Sorvall | 75253839 | |
| Adapters | Sorvall | 75003723 | |
| Rat anti-mouse CD16/CD32 | BD Biosciences | 553142 | Concentration: 0.5 – 1 μg/ 106 cells |
| Rat anti-mouse F4/80 | eBioscience | 17-4801 | Fluorophore conjugate: APC Concentration: 0.2 μg/ 106 cells Isotype control catalog number: 17-4321 |
| Rat anti-mouse CD11b | eBioscience | 11-0112 | Fluorophore conjugate: FITC Concentration: 0.5 μg/ 106 cells Isotype control catalog number: 11/1/4031 |
| Armenian Hamster anti-mouse CD11c | eBioscience | 12-0114 | Fluorophore conjugate: PE Concentration: 0.8 μg/ 106 cells Isotype control catalog number: Dec-88 |
| Goat anti-mouse MGL1/2 | R&D Systems | FAB4297P | Fluorophore conjugate: PE Concentration: 0.1 μg/ 106 cells Isotype control catalog number: IC108P |
| Goat anti-mouse CD206 | R&D Systems | FAB2535P | Fluorophore conjugate: PE Concentration: 0.1 μg/ 106 cells Isotype control catalog number: IC108P |
| Rat anti-mouse CCR2 | R&D Systems | FAB5538P | Fluorophore conjugate: PE Concentration: 0.1 μg/ 106 cells Isotype control catalog number: IC013P |
| Armenian Hamster anti-mouse TCRβ | BD Biosciences | 553174 | Fluorophore conjugate: APC Concentration: 0.2 μg/ 106 cells Isotype control catalog number: 553956 |
| Rat anti-mouse CD8a | BD Biosciences | 552877 | Fluorophore conjugate: PE-Cy7 Concentration: 0.8 μg/ 106 cells Isotype control catalog number: 552784 |
| Rat anti-mouse CD4 | BD Biosciences | 557956 | Fluorophore conjugate: Alexa Fluor 700 Concentration: 0.2 μg/ 106 cells Isotype control catalog number: 557963 |
Table 1. List of Materials and Reagents.
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