Måling granulocyt og monocyt Fagocytose og Oxidativ Burst Aktivitet i humant blod
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
Den granulocyt og monocyt fagocytose / oxidative udbrud (OB) aktivitetsanalyse en enkel, ligetil teknik, der ofte anvendes til at indsamle oplysninger om medfødte immunfunktion efter længere tids og intensiv træning. 1-4 Når man undersøger reaktion af immunsystemet til en stimulus, granulocytter og monocytter er af særlig interesse, fordi de spiller en central rolle i værtsforsvar og er de første immunceller at akkumuleres ved infektionsstedet. 5 Neutrofiler, en type granulocyt, er de første celler til at translokere ind beskadigede muskel væv efter intensiv træning. 6 Fagocytose og OB aktivitet er de mest almindelige mål for granulocyt og monocyt funktion, 7 og foretrækkes som indikatorer for medfødte immunforsvar celle funktion på grund af deres centrale rolle i både infektionen proces og reparationsprocessen.
Den i dette manuskript Uti assayZES en grundlæggende flowcytometer at tilvejebringe en kvantitativ bestemmelse af granulocyt og monocyt fagocytose og OB-aktivitet i fuldblod. Anvendelsen af helblod i denne teknik er fordelagtig, fordi den tillader assayet skal udføres uden tidskrævende oprensningsprocedure. Dette assay kan let modificeres til at rumme en række forskningsdesigns og lab kapaciteter. For eksempel, Liao et al. Anvendt en lignende teknik til at vise, at neutrofil OB aktivitet forøges, og neutrofil fagocytose formindskes efter traumatisk hjerneskade. 8 I et andet eksempel, McFarlin et al. Beskrev et elegant modifikation af denne teknik, der bruger allophycocyanin (APC ) -konjugeret CD66b og højtydende tandem APC-konjugeret CD45 mærkning med billedbaseret flowcytometri at klassificere granulocytter i form af deres fagocytose og OB aktiviteter. 7,9
Vores forskergruppe har brugt forskellige tilpasninger af denne technique som indikator for medfødt immunforsvar hos overvægtige, svært overvægtige, og atletisk befolkninger i næsten 20 år. 10,11 Teksten nedenfor vil give læseren trin-for-trin instruktioner til at udføre denne analyse og fremhæve områder, som læseren kan tilpasse at opfylde behovene i deres forskning.
Protocol
Representative Results
Discussion
Dette håndskrift giver en trin-for-trin-protokol til bestemmelse af to indikatorer for granulocyt og monocyt funktion. Vi har identificeret et par trin, der er afgørende for succes i denne analyse. Et sådant trin er isbadet inkubation, der forekommer umiddelbart før tilsætningen af bakterier. Grundig afkøling af alle prøverne vil minimere virkningen af temperatur på fagocytose og OB-aktivitet. Et andet kritisk trin er tilsætning af bakterier til hver prøve. A 8: 1 bakterier: fagocyt forholdet produ…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne anerkende Zack Shue, Casey John, og Lynn Cialdella-Kam for deres bistand i optimeringen af denne procedure.
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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