Medição de granulócitos e monócitos fagocitose e oxidativo Atividade Burst no Sangue
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
A explosão de granulócitos e monócitos fagocitose / oxidativo (OB) ensaio da atividade é uma técnica simples e direta que é frequentemente usado para reunir informações sobre a função imunológica inata após o exercício prolongado e intenso. 1-4 Ao estudar a resposta do sistema imunológico a um estímulo, granulócitos e monócitos são de particular interesse porque eles desempenham um papel central na defesa do hospedeiro e são as primeiras células do sistema imunológico a se acumular no local de infecção. 5 neutrófilos, um tipo de granulócitos, são as primeiras células de se translocar para o músculo danificado tecidos após o exercício intenso. 6 fagocitose ea atividade de OB são as medidas mais comuns da função de granulócitos e monócitos, 7 e são preferidos como indicadores da função celular imune inato por causa de seu papel central, tanto no processo de infecção e o processo de reparação.
O ensaio descrito no presente Utili manuscritozes um citómetro de fluxo de base para proporcionar uma determinação quantitativa da fagocitose de granulócitos e monócitos e atividade de OB no sangue total. O uso de sangue inteiro nesta técnica é vantajosa porque permite o ensaio a ser conduzido sem um procedimento de purificação que consomem tempo. Este ensaio pode ser facilmente modificado para acomodar uma variedade de modelos de investigação e de capacidades de laboratório. Por exemplo, Liao et al., Utilizou uma técnica semelhante para demonstrar que a actividade dos neutrófilos OB é aumentada e neutrófilos fagocitose é diminuída após lesão cerebral traumática. 8 Num outro exemplo, McFarlin et al. Descrevem uma modificação elegante desta técnica que utiliza aloficocianina (APC ) -conjugated CD66b e de alto desempenho conjunto rotulagem CD45 APC conjugada com a citometria de fluxo baseada em imagem para classificar os granulócitos em termos de suas atividades fagocitose e OB. 7,9
Nosso grupo de pesquisa tem usado várias adaptações desta technique como um indicador da função imune inata em populações com sobrepeso, obesas, e atléticas por quase 20 anos. 10,11 O texto abaixo irá fornecer o leitor com instruções passo-a-passo para a realização deste ensaio e destacar as áreas que o leitor possa se adaptar para atender às necessidades de suas pesquisas.
Protocol
Representative Results
Discussion
Este manuscrito fornece um protocolo passo-a-passo para a determinação dos dois indicadores de granulócitos e monócitos. Foram identificadas algumas etapas que são críticos para o sucesso deste ensaio. Um tal passo é a incubação em banho de gelo que ocorre imediatamente antes da adição de bactérias. arrefecimento exaustiva de todas as amostras irá minimizar o efeito da temperatura sobre a actividade de fagocitose e OB. Um outro passo crucial é a adição de bactérias para cada amostra. A 8: 1 bactérias: …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Os autores gostariam de reconhecer Zack Shue, Casey John e Lynn Cialdella-Kam para a sua assistência durante a otimização deste processo.
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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