ヒト血液中の測定顆粒球と単球食作用および酸化バースト活動
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
顆粒球及び単球貪食/酸化的バースト（OB）活性アッセイ頻繁に長時間の、集中運動後の先天性免疫機能に関する情報を収集するために使用されるシンプルな、ストレートフォワードの手法である。1-4に対する免疫系の応答を研究するときこれらは宿主防御において中心的な役割を果たし、感染部位に蓄積するための第一の免疫細胞であるため、刺激、顆粒球および単球は、特に重要である。5好中球、顆粒球の種類は、損傷した筋肉内に移行する最初の細胞であります激しい運動後の組織。6食作用およびOBの活動は、顆粒球や単球機能、7の最も一般的な尺度であり、なぜなら、感染プロセスと修復プロセスの両方におけるそれらの中心的役割の自然免疫細胞機能の指標として好ましいです。
この原稿utiliに記載のアッセイ全血における顆粒球や単球食作用およびOBの活動の定量的な決意を提供するために、サイトメーターの基本的な流れをZES。それは、アッセイは、時間のかかる精製操作なしで行われることを可能にするため、この技術では全血を使用することが有利です。このアッセイは、容易に研究設計および実験室機能の様々に適応するように修正することができます。例えば、リャオらは 、その好中球OB活性を示すために同様の技術を利用したが、外傷性脳損傷後に増加し、好中球の食作用低下する。8の別の実施例では、McFarlin らは 、アロ使用するこの技術のエレガントな修飾（APC記載しました）それらの食作用およびOB活動に関して顆粒球を分類するCD66bと画像ベースのフローサイトメトリーと高性能タンデムAPC共役CD45標識を抱合。7,9
私たちの研究グループは、このテの様々な適応を使用していますほぼ20年間、太りすぎ肥満、および運動競技集団における先天性免疫機能の指標としてchnique。10,11のテキストは、以下のこのアッセイを実行するためのステップバイステップの手順を読者に提供し、読者が適応させることができる領域を強調表示します彼らの研究のニーズを満たすことができます。
Protocol
Representative Results
Discussion
この原稿は、顆粒球と単球機能の2つのインジケータの決意のためのステップバイステップのプロトコルを提供します。私たちは、このアッセイの成功に不可欠ないくつかの手順を確認しています。そのような工程は、細菌の添加の直前に発生した氷浴インキュベーションです。すべてのサンプルの徹底的な冷却は、食作用およびOB活性に対する温度の影響を最小限に抑えることができま?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
著者は、この手順の最適化中に彼らの援助のためにザック・シュー、ケーシー・ジョン、およびリンCialdella-カムを承認したいと思います。
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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