一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血
Summary
Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.
Abstract
单核细胞是可通过与某些慢性炎性疾病相关的病原体和炎症被激活先天免疫细胞。单核细胞激活诱导效应职能和免受氧化糖酵解来代谢随之而来的转变，伴随增加葡萄糖转运蛋白表达。这增加了糖酵解代谢也为单核细胞经过培训免疫力，先天免疫记忆形式观察。虽然在体外单核细胞检查葡萄糖转运蛋白表达和葡萄糖摄取的协议进行了说明，没有已被多参数流式细胞仪在全血检测。我们描述了荧光葡萄糖类似物2-NBDG摄取全血中总单核细胞和经典（CD14 + CD16 – ）测定的多参数流式细胞协议，中间体（CD14 + CD16 +）和非经典（ CD14 + CD16 +）单核细胞亚群。该方法可用于检查稳态和炎症性疾病中总单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和葡萄糖摄取，并且可以容易地修改，以检查血液中的其它白细胞和白细胞亚群的葡萄糖摄取。
Introduction
单核细胞是正在迅速动员到感染和炎症1的位点的人的先天免疫系统的重要组成部分。单核细胞的活化是限制由病原体产生急性损害的关键，也是中央对多种慢性疾病，包括动脉粥样硬化2，3癌症和HIV 4,5的发病机制。
静息和活化的单核细胞的代谢显着不同，以利用氧化代谢利用糖酵解代谢（ 即 ，葡萄糖发酵成乳酸）静息单核细胞和活化的单核6。单核细胞的活化诱导葡萄糖转运，可以增加葡萄糖摄取糖酵解代谢7的表达。单核细胞葡萄糖转运蛋白1（的Glut1）是激活期间上调一个这样的转运，其表达已显示导致生产v中促炎细胞因子的itro和在肥胖小鼠8的脂肪组织。由卡波西肉瘤相关疱疹病毒单核细胞系感染导致9的Glut1细胞上调，我们最近发现，慢性感染艾滋病毒期间的Glut1表达的单核细胞百分比增加是未经处理及联合抗逆转录病毒疗法治疗的感染10时在座。总而言之，这些研究表明，葡萄糖摄取和由单核细胞糖酵解代谢许多炎性疾病的重要方面。因此，一个简单的方法来测量稳态期间的单核细胞的Glut1表达和葡萄糖摄取和炎性疾病可能是使用了广泛的研究人员。
人单核细胞是异质的，正在由能够由细胞表面标记物CD14和CD16 11,12的差异表达进行检查三个不同的子集。经典单核细胞表达CD14的较高水平，但不表达CD16（CD14 + CD16 – ），中间的单核细胞表达CD14的高水平和CD16（CD14 + CD16 +）的中间电平，和非经典的单核细胞表达CD14的水平低和CD16高的水平（CD14 + CD16 +）。表达CD16的单核细胞被称为CD16 +单核细胞，这相比CD16 –单核细胞有炎性细胞因子的高表达和能力，更有效地呈递抗原13,14。单核细胞的大约10％与炎症15中观察到的更高的百分比的动态平衡期间表达CD16。单核细胞亚群与某些疾病状态相关联，并且可能是疾病和疾病进展16的有用的生物标志物。
我们的目标是确定可以测量通过在尽可能接近的phy条件人单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和葡萄糖摄取的方法siological条件越好。以前的研究测量的单核细胞葡萄糖转运蛋白的表达和葡萄糖摄取17,18，虽然这些检测方法可以比较生理条件19已经改变蛋白表达分离的单核细胞，并没有以前的研究调查了人单核细胞亚群。使用多参数流式细胞术，我们描述的方法来检查该荧光葡萄糖类似物2-NBDG按总单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和摄取（基于CD14和CD16的表达）全未经处理的血液内。
Protocol
Representative Results
Discussion
这里描述的协议细节的简单方法来检查在全血中的单核细胞和单核细胞亚群的葡萄糖转运蛋白表达和荧光葡萄糖类似物的摄取。通过在全血评估-2- NBDG摄取，此技术允许类似于在体内条件。先前的研究中通过密度离心17日全血中分离的单核细胞检测6 NBDG摄取。然而，这项研究并没有审查的单核细胞亚群，并从全血中单核细胞的分离可以潜在地改变某些细胞表面分子19的表达。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由澳大利亚中心艾滋病毒和肝炎病毒研究从华盛顿艾滋病研究中心大学（CFAR），根据奖号AI027757美国国立卫生研究院资助的程序，它支持（ACH 2）和2010年的发展拨款（CNIHR）资助通过下面的NIH研究所和研究中心（NIAID，NCI，镍氢电池，NIDA，NICHD，NHLBI，NIA）。 CSP是CNIHR和ACH 的赠款获得者。 SMC是澳大利亚（NHMRC）主要研究奖学金的国家健康和医学研究理事会的收件人。作者非常感谢由伯纳特机构收到了维多利亚经营性基础设施项目的支持这项工作的贡献。我们承认格扎Paukovic和伊娃ORLOWSKI – 奥利弗从AMREP流式细胞仪核心设施流式细胞仪培训和技术咨询协助。我们感谢安格斯摩根媒体教练和视频拍摄的组织。我们的感激杰西马森和讨伐异教徒阿卜杜勒阿齐兹K. Alzahrani在视频拍摄过程中的实验室协助。我们感谢大卫博士SIMAR在医学科学，新南威尔士大学，澳大利亚谁提供关键的方法建议，学校的努力。 CSP想感谢www.nice-consultants.com用于图形磋商。
作者的贡献：
CSP构思项目，设计并进行了实验，分析和解释数据，并且写了稿子。 JJA解释数据和撰写文章。 TRB写的稿子。 JMM解释的数据，提出关键知识的建议，并审查了原稿。 SMC解释的数据，做出关键的智力建议并审查了原稿。
Materials
| VACUETT Tube 9 ml ACD-B anticoagulant tubes | Greiner Bio-One GmbH | 455094 | |
| 5 ml sterile polypropylene tubes | BD Biosciences | 352063 | |
| Albumin from Bovine Serum (BSA) | Sigma-Aldrich | A7906 | |
| 16% formaldehyde solution | Electron Microscopy Science | 15710 | |
| BD FACS lysing solution (10X) | BD Biosciences | 349202 | Dilute BD FACS lysing solution 1/10 with deionized water for working concentration (store for up to 1 week at 4°C) |
| anti-CD3-PE | BD Biosciences | 555340 | |
| anti CD14-APC | BD Biosciences | 555399 | |
| anti-CD16-PECy7 | BD Biosciences | 557744 | |
| anti-Glut1-FITC | R & D Systems | FAB1418F | |
| IgG2b-FITC | R & D Systems | IC0041F | |
| 2-NBDG | Life technologies | N13195 | Suspend 5 mg of 2-NBDG into 1 ml of deionized water to make a 14.60 mM stock solution (keep for up to 6 months at 4°C). To make the working 2-NBDG concentration, dilute stock 1/100 with 1X DPBS. Cover with foil. (store for up to 1 week at 4°C) |
| Dulbecco’s Phosphate Buffered Saline (1X) | Life technologies | 14190-144 | To make wash solution, add 0.5 g BSA per 100 ml DPBS (store for up to 2 weeks at 4°C) |
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