Summary

脂肪组织免疫细胞的分离

Published: May 22, 2013
doi:

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 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

1。试剂和耗材发起这个实验协议之前,需准备以下试剂: 70%乙醇 1X PBS 1X的DPBS(无钙,镁)与0.5%牛血清白蛋白补充 FACS缓冲液:1X DPBS(无钙,镁),2mM的EDTA,1%FCS的 ACK缓冲液:150mM的氯化铵的10mM KHCO 3,和0.1mM EDTA在水中的Na 2 2。脂肪组织的收获和准备安乐死老鼠IACUC批准程序?…

Representative Results

雄性小鼠附睾脂肪垫的AT胶原酶消化后差速离心法分离的SVF喂食低脂肪(10%千卡来自脂肪)或高脂肪(60%千卡来自脂肪)饮食(LFD和HFD ),分别为16周。然后SVF细胞标记的荧光团标记的第一抗体,通过FACS分析可行的自动取款机( 图1)和AT&T的细胞( 图2)的量化比例。 图1A和2A中描绘了初始的门控,包括光散射,双重歧视和生存能力门。应该?…

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.

References

  1. Rodbell, M. Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis. The Journal of Biological Chemistry. 239, 375-380 (1964).
  2. Danse, L. H., Verschuren, P. M. Fish oil-induced yellow fat disease in rats. III. Lipolysis in affected adipose tissue. Veterinary Pathology. 15, 544-548 (1978).
  3. Weisberg, S. P., et al. Obesity is associated with macrophage accumulation in adipose tissue. The Journal of Clinical Investigation. 112, 1796-1808 (2003).
  4. Xu, H., et al. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. The Journal of Clinical Investigation. 112, 1821-1830 (2003).
  5. Mathis, D., Shoelson, S. E. Immunometabolism: an emerging frontier. Nature Reviews. Immunology. 11, 81 (2011).
  6. Stefanovic-Racic, M., et al. Dendritic cells promote macrophage infiltration and comprise a substantial proportion of obesity-associated increases in CD11c+ cells in adipose tissue and liver. Diabetes. 61, 2330-2339 (2012).
  7. Liu, J., et al. Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice. Nature Medicine. 15, 940-945 (2009).
  8. Feuerer, M., et al. Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nature Medicine. 15, 930-939 (2009).
  9. Nishimura, S., et al. CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity. Nature Medicine. 15, 914-920 (2009).
  10. Winer, S., et al. Normalization of obesity-associated insulin resistance through immunotherapy. Nature Medicine. 15, 921-929 (2009).
  11. Winer, D. A., et al. B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies. Nature Medicine. 17, 610-617 (2011).
  12. Wu, L., et al. Activation of invariant natural killer T cells by lipid excess promotes tissue inflammation, insulin resistance, and hepatic steatosis in obese mice. Proceedings of the National Academy of Sciences of the United States of America. 109. , 1143-1152 (2012).
  13. Wu, D., et al. Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science. 332, 243-247 (2011).
  14. Elgazar-Carmon, V., Rudich, A., Hadad, N., Levy, R. Neutrophils transiently infiltrate intra-abdominal fat early in the course of high-fat feeding. Journal of Lipid Research. 49, 1894-1903 (2008).
  15. Talukdar, S., et al. Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase. Nature Medicine. 18, 1407-1412 (2012).
  16. Hagman, D. K., et al. Characterizing and quantifying leukocyte populations in human adipose tissue: impact of enzymatic tissue processing. Journal of Immunological Methods. 386, 50-59 (2012).
  17. Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J. Vis. Exp. (41), e1546 (2010).
  18. Kotecha, N., Krutzik, P. O., Irish, J. M. Web-based analysis and publication of flow cytometry experiments. Current Protocols in Cytometry. Chapter 10, Unit 10 (2010).
  19. Cho, C. H., et al. Angiogenic role of LYVE-1-positive macrophages in adipose tissue. Circulation Research. 100, e47-e57 (2007).
  20. Roederer, M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry. 45, 194-205 (2001).

Play Video

Cite This Article
Orr, J. S., Kennedy, A. J., Hasty, A. H. Isolation of Adipose Tissue Immune Cells. J. Vis. Exp. (75), e50707, doi:10.3791/50707 (2013).

View Video