在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估
Summary
This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.
Abstract
本文从小型全血样本中的白细胞快速，标准化富集的协议描述。这个过程是基于红细胞的低渗裂解和可以应用到人类的样本以及非人类来源的血液。的约50至100微升小初始样品体积使得这种方法适用于反复采血从小型实验室动物。此外，白细胞富集分钟之内，并与有关化学品和仪器仪表材料低的努力所取得，使得这种方法适用于多个实验室环境。
白细胞的标准化纯化是用高选择性染色法来评价血红素过氧化物酶的卤化过氧化物酶，髓过氧化物酶（MPO）和嗜酸性粒细胞过氧化物酶（EPO）， 即，次氯酸和次溴酸（次氯酸和次溴酸）的形成相结合。虽然MPO是NEUT强烈表达rophils，在人血液中，以及在单核细胞中最丰富的免疫细胞类型，相关的酶的EPO是专门在嗜酸性粒细胞中表达。这些酶的卤化活性是通过使用几乎HOCl-和次溴酸特异性染料氨基苯基荧光素（APF）和主过氧化物过氧化衬底氢处理。在随后的流式细胞分析所有过氧化物酶阳性细胞（嗜中性粒细胞，单核细胞，嗜酸性粒细胞）的区分和它们的卤化过氧化物酶的活性可以被量化。因为APF染色可以与细胞表面标记物的应用相结合，这个协议可以扩展到专门处理白细胞亚级分。该方法适用于同时检测在人类和啮齿动物白细胞次氯酸和次溴酸的生产。
鉴于这些酶产品在慢性炎症性疾病的广泛和多样讨论免疫的作用，这个协议可能有助于更好地理解免疫白细胞衍生的血红素的过氧化物酶的相关性。
Introduction
多形核白细胞（PMN，也称为粒细胞）和单核细胞代表在血液中1,2-先天免疫系统的重要细胞成分。它们向主防御病原体以及所获取的免疫系统的活化和全身性的炎症反应2-4的启动。然而尤其嗜中性粒细胞，最丰富的类型粒，和单核细胞也显著有助于急性炎性事件5的调节和终止。因此，这些细胞也可能在慢性炎性疾病如类风湿关节炎6,7-重要作用。事实上，哮喘，慢性炎性气道疾病，特点是嗜酸性粒细胞的削弱的细胞凋亡，在血液8的第二大粒型。然而粒细胞凋亡及快速拆卸由巨噬细胞是细胞终止在两个基本步骤炎症9-11。
在已命名的免疫细胞两个密切相关的酶，即髓过氧化物酶（MPO，中性粒细胞和单核细胞）和嗜酸性粒细胞过氧化物酶（EPO，嗜酸性粒细胞）可以发现12,13。这些血红素过氧化物酶古典相关，因为它们两个电子氧化（伪）卤化为相应的低（伪），其为它们的杀菌性能14-16已知亚卤酸体液免疫应答。在生理条件下的MPO主要形式次氯酸（次氯酸）和hypothiocyanite –而后者和次溴酸（次溴酸）由EPO 17-19形成（OSCN）。新的结果表明，这种（伪）卤化酶活性也可能有助于炎性反应的调节和免疫反应20,21的终止。事实上，次氯酸生产由MPO和衍生产品被证明抑制Ť基于细胞的适应性免疫应答22-24。
为了在慢性炎症性疾病，以获得从先天免疫系统更见解白细胞的免疫作用，并确定MPO和EPO的贡献这种生理功能，我们开发迅速丰富从小血样白细胞用于随后特定的方法测定这些细胞中的卤化过氧化物酶活性。为红细胞耗尽我们选择的标准方法，包括用蒸馏水，这导致了快速的白细胞富集在低材料成本的两后续低渗裂解步骤。对于卤化MPO随后的决心和EPO活动HOCl-和次溴酸专用染料荧光氨苯（APF）用于25-27。在对比的非特异性过氧化物酶染色方法28,29中的应用，这种方法使卤化过氧化物酶活性的选择性检测，这往往是在严重INFL障ammation 30,31。
Protocol
从健康志愿者获得了人类所有的血液样本，并应用白细胞浓缩协议遵循莱比锡大学医学院伦理委员会的指导方针。与大鼠血液实验是由负责当地的伦理委员会（Landesdirektion萨克森，Referat 24），根据动物护理和使用指南德国批准。 1.实验装置注意：作为用于从血液样品的红细胞的枯竭的低渗裂解过程是时间关键的步骤中，该协议的这部分预先准备所有必要?…
Representative Results
如先前报道上述方法原来是既适用于人类和非人类材料32。此外如图用于与哮喘症状的小鼠将APF染色可检测的全身促炎状态的差异的合适的工具。因此，在我们使用该协议随后的研究中反复雌性黑刺鼠大鼠降植烷诱导关节炎（PIA）评估MPO（和EPO）的卤化活动。白细胞富集和该实验期间进行染色的代表性实例示于图1。在麻醉下从动物的眼球后静脉…
Discussion
嗜中性粒细胞是人体血液中最丰富的白细胞的过氧化物酶阳性细胞的分离通常只着重于这些细胞，并包括由密度梯度离心38从其它白细胞嗜中性粒细胞的分离。然而，作为嗜中性粒细胞是鼠的血液样本中少得多的丰富39对后者更复杂的方法，必须使用40。而且这两种方法也导致除去从样品中的过氧化物酶阳性单核细胞的和，由于需要更大的血液体积（ 例如，400微升<su…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was made possible by funding from the German Federal Ministry of Education and Research (BMBF, 1315883) as well as by the Sächsische Aufbaubank (SAB) project 100116526 from a funding of the European Regional Development Fund (ERDF).
Materials
| materials/equipment | |||
| 15 ml centrifugation tubes | VWR/Corning | 734-0451 | – |
| 1.5 ml sample tubes | VWR/Eppendorf | 211-2130DE | – |
| Pipettes for volumes up to 5 ml | Eppendorf | e.g. 3120000070 | We are using Eppendorf Resarch plus pipettes with adjustable volumes in the range 1-10 µl, 10-100µl, 100-1000 µl an 500-5000µl |
| laminar flow bench | Thermo Electron Corperation | HeraSafe | – |
| Vortex mixer | Bender & Hobein AG | Vortex Genie 2 | – |
| Tabletop centrifuge | Kendro Laboratory Products | Laborfuge 400R | The centrifuge should be able to be used at 450x g |
| Small centrifuge | eppendorf | 5415D | The centrifuge should be able to be used at 400x g |
| Incubator | Heraeus | cytoperm 2 | Settings: 37 °C, 95% humidity, 5 % CO2 content |
| UV-Vis spectrophotometer | Varian | Cary 50 bio | A spectrum between 200 and 300 nm has to be recorded. Thus quartz cuvettes have to be applied |
| Flow cytometer | Becton, Dickinson | BD Facs Calibur | Any flow cytometer can be used which is equiped with a laser suitable for the excitation of fluorescein (e.g 488 nm argon laser) |
| Name | Company | Catalog Number | Comments |
| Chemicals | |||
| Phosphate buffered saline (PBS) | amresco | K812 | sterile solution, ready to use |
| Sigma-Aldrich | P4417 | tablets for solving in 200 ml millipore water | |
| Hanks balanced salt solution (HBSS) with Ca(2+) | Sigma-Aldrich | H1387 | 970 mg/100 ml, carefully check and adjust the pH value to 7.4 |
| Hydrochloric acid | Merck Millipore | 1.09057.1000 | 1M solution |
| Sodium hydroxide | Riedel-deHaën | 30620 | Solid pellets. For a 1M solution solve 4 g/100 ml Millipore water |
| Aminophenyl fluorescein | Cayman | 10157 | 5 mg/ml solution (11.81 mM) in methyl acetate, aliquotes of e.g. 100 µl should be prepared and stored at -20 °C |
| Hydrogen peroxide | Sigma-Aldrich | H1009 | This 30% stock solution corresponds to a concentration of about 8.8 M. Further dilutions have to be freshly prepared in distilled water immediately prior to use and quantified by absorbance measurements |
| 4-aminobenzoic acid hydrazide (4-ABAH) | Sigma-Aldrich | A41909 | A first stock solution of 1 M should be prepared in DMSO a second one of 100 mM by 1:10 dilution in HBSS |
| DMSO | VWR chemicals | 23500.26 | – |
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Citazione di questo articolo
Flemmig, J., Schwarz, P., Bäcker, I., Leichsenring, A., Lange, F., Arnhold, J. Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples. J. Vis. Exp. (113), e54484, doi:10.3791/54484 (2016).