人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动
Summary
The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.
Abstract
The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots (“H” and “F”) of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.
Introduction
粒和单核细胞的吞噬作用/氧化爆发（OB）活性测定是经常用来收集关于以下长时间和密集运动先天性免疫功能信息的简单，直接的技术。1-4当研究免疫系统的反应一种刺激，粒细胞和单核细胞是特别感兴趣的，因为它们在宿主发挥防御核心作用，并且是第一个免疫细胞在感染部位蓄积。5嗜中性粒细胞，一式粒，是第一细胞移位到受损肌肉组织下面的剧烈运动。6吞噬和OB活性的粒细胞和单核细胞的功能，7的最常见的措施和优选作为因为它们在两个感染过程和修复过程中心作用先天免疫细胞功能的指标。
在这个手稿utili描述的测定ZES一个基本的流式细胞仪提供全血粒细胞和单核细胞吞噬功能和OB活动的定量测定。在此技术中使用的全血是有利的，因为它允许在不一项费时的纯化方法来进行测定。该测定可以很容易地进行修改，以适应各种研究设计和实验室能力。例如，Liao等利用类似的技术，以显示该嗜中性粒细胞的OB活性增加和中性粒细胞的吞噬作用降低创伤性脑损伤。8在另一实例中，麦克法林等人描述该技术，使用别藻蓝蛋白的优雅修改（APC ）结合的CD66b和高性能串联APC标记的CD45标记与基于图像的流式细胞仪在其吞噬作用和OB活动方面分类粒细胞。-7,9-
我们的研究小组已用这种TE的各种改编chnique如超重，肥胖和运动人群先天免疫功能，为近20年来的一个指标。10,11文字下面将提供一步一步的指导读者进行这种测定，并强调指出，读者可以适应领域以满足他们的研究的需要。
Protocol
Representative Results
Discussion
这份手稿提供了一个一步一步的协议粒细胞和单核细胞功能的两个指标的决心。我们已经确定了对这个实验成功的关键几步之遥。一个这样的步骤是发生之前立即加入细菌的冰浴温育。所有样品的彻底冷却将最大限度地减少温度对吞噬和OB活性的作用。另一个关键步骤是在加入细菌到各样品中。 A 8：1的细菌：巨噬细胞产生比最佳效果;然而其他研究人员可能会发现，这个比例将需要进行修改，以?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者在此过程中的优化过程中承认扎克树，约翰·卡西和林恩Cialdella鉴的协助。
Materials
| 12 x 75 mm tubes, with cap | VWR | 20170-579 | You will need 2 tubes per sample ("H" and "F") plus 3 tubes per batch (for assay controls; "F", "H", and "Blood only"). |
| PBS (10X), pH 7.4 | Life Technologies | 70011-044 | |
| Bottle top 0.2 µm cellulose acetate filter (500 ml capacity) | Fisher Scientific | 09-741-07 | |
| Glucose, powder | Life Technologies | 15023-021 | |
| Citric acid (anhydrous, cell culture tested, plant cell culture tested) | Sigma-Aldrich | C2404-100G | |
| Sodium citrate tribasic dihydrate | Sigma-Aldrich | S4641-25G | |
| Dihydroethidium (Hydroethidine) (HE) | Life Technologies | D11347 | |
| Dimethyl sulfoxide (DMSO) Anhydrous | Life Technologies | D12345 | |
| Staphylococcus aureus (Wood strain without protein A) BioParticles, fluorescein conjugate (FITC) | Life Technologies | S2851 | |
| Staphylococcus aureus (Wood strain without protein A) BioParticles, unlabeled | Life Technologies | S-2859 | |
| Trypan Blue Solution, 0.4% | Life Technologies | 15250-061 | |
| 4.0 mL vacutainer containing 7.2 mg K2EDTA, spray-dried | VWR | BD367861 | You will need 1 K2 EDTA blood collection tube per sample. |
| 4.0 mL vacutainer containing 68 USP units Lithium Heparin, spray-coated | VWR | BD367884 | You will need 1 Lithium Heparin blood collection tube per sample. |
| COULTER Ac·T 5diff CP | Beckman Coulter | 6605705 | i.e., hematology analyzer |
| COULTER Ac·T 5diff Rinse | Beckman Coulter | 8547167 | |
| COULTER Ac·T 5diff Fix | Beckman Coulter | 8547171 | |
| COULTER Ac·T 5diff WBC Lyse | Beckman Coulter | 8547170 | |
| COULTER Ac·T 5diff Hgb Lyse | Beckman Coulter | 8547168 | |
| COULTER Ac·T 5diff Cal Calibrator | Beckman Coulter | 7547175 | |
| COULTER Ac·T 5diff Control Plus | Beckman Coulter | 7547198 | |
| AcT 5diff Diluent | Beckman Coulter | 8547169 | |
| 200 µL extended-length pipette tips | VWR | 37001-526 | |
| Insulated ice pan | VWR | 89198-980 | For ice water bath. |
| Open metal tube rack | VWR | 60916-702 | |
| Fetal Bovine Serum | Life Technologies | 26140-111 | |
| TQ-Prep Workstation | Beckman Coulter | 6605429 | i.e., automated cell lyse preparation workstation |
| ImmunoPrep Reagent System | Beckman Coulter | 7546999 | i.e., RBC lyse/WBC fix reagent system |
| Cytomics FC500 MCL Flow Cytometry System with CXP Software | Beckman Coulter | 626553 | |
| IsoFlow Sheath Fluid | Beckman Coulter | 8547008 | |
| COULTER CLENZ | Beckman Coulter | 8546929 | |
| Flow-Check Fluorospheres | Beckman Coulter | 6605359 | i.e., alignment and fluidics verification fluorospheres |
| FlowJo Software | FlowJo | FlowJo | i.e., flow cytometry analysis software |
| Excel Software | Microsoft | Microsoft Excel | i.e., electronic spreadsheet program |
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