利用共焦显微镜可视化巨噬细胞胞外陷阱
Summary
巨噬细胞胞外陷阱是一个新描述的实体。这篇文章将集中于共焦显微镜方法和如何他们是可视化的体外和体内从肺部样本。
Abstract
一种用于确定中性粒细胞胞外陷阱 (网) 存在的主要方法是共焦显微镜。我们已经修改了建立的共焦显微镜方法来可视化巨噬细胞胞外陷阱 (纽约大都会)。这些胞外陷阱的定义是存在的胞外染色质与表达的其他成分, 如颗粒蛋白酶, 氨组蛋白, 和肽酸 deiminase (PAD)。一般在接触刺激后测量, 与 un-stimulated 样本相比, 大都会的表达。样品也包括背景和同种控制。使用定义良好的图像分析软件分析单元。共聚焦显微镜可用于定义的存在的两个体外和在体内肺组织。
Introduction
中性粒细胞胞外陷阱 (网), 首先由 Brinkmann et al.描述1它们主要是针对感染 (特别是细菌) 而产生的, 在主机防御中具有重要作用12。他们也被描述为对非传染性疾病, 包括血管炎和系统性红斑狼疮 (SLE) 的反应发生;并对丝裂原佛波 12-myrisate 13 乙酸 (PMA)2,3。最近已经认识到, 其他细胞类型也可能产生胞外陷阱, 包括巨噬细胞。巨噬细胞胞外陷阱 (纽约大都会) 在文献4,5中还不是一个定义良好的实体。我们最近建立的方法, 以检测的存在, 两个在体外和在体内6,7。本文将介绍用共焦显微镜测量大都会的方法。
与其他细胞通路 (如细胞凋亡8) 相区别的 NETosis 的主要特征是染色质的挤压与: (1) 组蛋白的 citrullination (H3Cit)9, (2) 颗粒蛋白酶的表达,10和 (3) 参与肽精氨酸 deiminase (PAD) 411,12。巨噬细胞也表达 H3Cit, 颗粒蛋白酶, 和垫, 这些功能可以用来定义的存在, 大都会。
大都会在肺中具有特别重要的作用, 因为巨噬细胞是肺肺泡和气管中的主要干细胞, 在引导细胞免疫应答感染/炎症方面起着最初的作用。此外, 虽然肺的大部分是空的空间 (例如, 在肺泡内), 大都会有可能扩展到可用的空间, 与固体器官形成对比。
最广泛使用的方法来定义存在的网是通过共焦显微镜。目前还没有一个明确的方法来衡量纽约大都会队。测量网的技术被适应了测量在这个协议的存在的大都会。该方法的主要要求是获得共焦显微镜和适当的成像软件进行分析。
Protocol
本协议遵循以下的实验: (1) 人类由蒙纳士医学中心伦理委员会和 (2) 动物的伦理委员会的墨尔本大学. 1. 支气管肺泡灌洗 (球) 巨噬细胞 使用球获得肺巨噬细胞: (1) 经支气管镜检查的人体受试者 6 , (2) 小鼠使用 intra-tracheal 吸入 13. 注: 巨噬细胞的获得通常会引起一些活化作用。巨噬细胞是从需要支气管镜检查, 以调查潜在的?…
Representative Results
可从球样, 肺组织和较厚的肺切片中, 3 维图像可视化。在图 1中显示了一个在一个球样中可视化的大都会示例。大都会的形态学根据其成熟阶段而变化。显微镜上第一个可探测的特征是细胞核在细胞边缘的运动。其次是胞外染色质与其他 co-expressed 介质, 如 H3Cit 和颗粒蛋白酶。在早期阶段, 细胞仍然有一个大致球形的形状与胞外陷阱表达。在以后的阶?…
Discussion
本评价中描述的方法基于用于定义网的存在14。巨噬细胞是到目前为止的主要单元类型在球样品和这种收集方法特别适合研究大都会。如果在球中存在红细胞, 则应使用氯化铵裂解这些细胞。该球的程序将一般激活巨噬细胞, 因此预计将有 un-stimulated 样本存在。巨噬细胞通常是非常粘附的。由于这些细胞是主要的细胞类型, 而且它们的形态也可以区分, 因此, 它们通常不需要特定的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作的经费来自蒙纳士大学、国家卫生和医学研究理事会以及蒙纳隆和睡眠研究所的赠款。作者要感谢在蒙纳士健康, 朱迪卡拉汉, 亚历克斯富尔切, Kirstin Elgass 的临床免疫学工作人员和蒙纳登 Lo 的微成像 (mmi) 的帮助与共聚焦显微镜成像, 和作者承认的 MMI 设施蒙纳士大学。作者承认的设施, 和科学和技术援助的蒙纳的组织学平台。
Materials
| Human samples: Primary antibodies | |||
| Rabbit anti-human H3Cit (Citrulline R26) | Abcam | AB5103 | 10 ug/ml |
| Rabbit anti-human MMP12 | Novus Biological | NB110-57214 | 1:100 concentration not quantifiable |
| Mouse anti-human MMP9 | Novus Biological | AB119906 | 1:200 ascites, no concentration given |
| Rabbit anti-human PADI2/PAD2 | Abcam | AB16478 | 7 ug/ml |
| Mouse anti-human PADI4/PAD4 | Abcam | AB128086 | 10.1 ug/ml |
| Sheep anti-human NE | LifeSpan Bioscience | LS-B4244 | 25 ug/ml |
| Name | Company | Catalog Number | Comments |
| Mouse samples: Primary antibodies | |||
| Goat anti-mouse MMP9 | Abcam | AF909 | 10 ug/ml |
| Rabbit anti-mouse H3Cit (Citrulline R26) | Abcam | AB5103 | 10 ug/ml |
| Rat anti-mouse F4/80 | In-house (hybridoma) | In house | 20 ug/ml |
| Sheep anti-human Anti-HNE /NE | Sapphire Bioscience | LS-B4244 | 25 ug/ml |
| Mouse anti-human PADI4/PAD4 | Abcam | AB128086 | 10.1 ug/ml |
| Super-frost plus slides | Menzel | S41104A | |
| Dapi-prolong gold | Molecular probes | P36931 | |
| Triton-X 100 | Sigma-Aldrich | 85111 | |
| Ammonium chloride | Sigma-Aldrich | E9434 | |
| Name | Company | Catalog Number | Comments |
| Secondary antibodies | |||
| Chicken anti-rabbit AF 488/ | Life technologies | A-21441 | |
| Chicken anti-rabbit AF 594 | Life technologies | A-21442 | |
| Chicken anti-goat AF 594 | Life technologies | a-21468 | |
| Chicken anti-mouse AF488 | Life technologies | A-21200 | |
| Donkey anti-sheep AF 594 | Life technologies | A-11018 | |
| Chicken anti-mouse AF 647 | Life technologies | A-21463 | |
| Donkey anti-sheep AF 594 | Life technologies | A-11016 | |
| Isotype control | |||
| Rabbit IgG | In house | ||
| Rabbit IgG | In house | ||
| Mouse IgG1 | BD Bioscience | 550878 | |
| Rabbit IgG | In house | ||
| Mouse IgG2a | BioLegend | 400201 | |
| Sheep IgG | In house | ||
| Name | Company | Catalog Number | Comments |
| Software Programs | |||
| Imaris | Bitplane | ||
| Image J | NIH | ||
| To average intensity of fluorphores a standard office application like Microsoft Excel can be used | |||
| Name | Company | Catalog Number | Comments |
| Microscopes | |||
| C1 Confocal scanning microscope | Nikon | ||
| FV1200 Confocal scanning microscope | Olympus | ||
| Name | Company | Catalog Number | Comments |
| Tissue sources | |||
| Human BAL samples from the bronchoscopy suite at Monash Medical Centre | |||
| Mouse BAL samples and lung tissue from the Department of Pharmacology, University of Melbourne. | |||
| Name | Company | Catalog Number | Comments |
| Media | |||
| RPMI | Sigma-Aldrich | r8758 | |
| Fetal calf serum | Sigma-Aldrich | F0926 | |
| L-glutamine | Sigma-Aldrich | G7513 | |
| Name | Company | Catalog Number | Comments |
| Other reagents | |||
| Sudan black | Sigma-Aldrich | 199664 | |
| paraformaldehyde/periodate/lysine (PLP) fixative | Sigma-Aldrich | 27387 | |
| Xylene | Sigma-Aldrich | 214736 | |
| Ketamine | Sigma-Aldrich | K2753 | |
| Natural formalin | Sigma-Aldrich | 42904 | |
| Paraffin | Sigma-Aldrich | 327204 | |
| Agarose | Sigma-Aldrich | A2576 | |
| Solvent 3B | Hi-Chem | 2026 | |
| Coverslips 12 ml | Sigma-Aldrich | S1815 | |
| Coverslips 60 x 24 ml | Sigma-Aldrich | C6875 | |
| Name | Company | Catalog Number | Comments |
| Mice | |||
| c57 black 6 | Monash animal research platform (MARP) | ||
| BALB/c | MARP |
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Citar este artigo
Sharma, R., O’Sullivan, K. M., Holdsworth, S. R., Bardin, P. G., King, P. T. Visualizing Macrophage Extracellular Traps Using Confocal Microscopy. J. Vis. Exp. (128), e56459, doi:10.3791/56459 (2017).