Immunology and Infection
Published: September 12th, 2016
The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.
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.
The granulocyte and monocyte phagocytosis/oxidative burst (OB) activity assay is a simple, straight-forward technique that is frequently used to gather information about innate immune function following prolonged and intensive exercise.1-4 When studying the response of the immune system to a stimulus, granulocytes and monocytes are of particular interest because they play a central role in host defense and are the first immune cells to accumulate at the site of infection.5 Neutrophils, a type of granulocyte, are the first cells to translocate into damaged muscle tissue following intensive exercise.6 Phagocytosis and OB activit....
NOTE: All blood collection procedures were conducted in accordance with the guidelines set forth by the Appalachian State University (ASU) Institutional Review Board (IRB).
1. Assay Preparation
Prolonged and intensive exercise has profound effects on innate immune function, including natural killer cell function, macrophage cytokine-mediated response to viral infection, and granulocyte and monocyte phagocytosis and OB activity. Multiple studies indicate that granulocyte and monocyte phagocytosis increases significantly post-exercise, reflecting the inflammation induced by muscle damage and metabolic demands. In contrast, granulocyte and monocyte OB activity decreases post-exerci.......
This manuscript provides a step-by-step protocol for the determination of two indicators of granulocyte and monocyte function. We have identified a few steps that are critical to the success of this assay. One such step is the ice-bath incubation that occurs immediately prior to the addition of bacteria. Thorough cooling of all samples will minimize the effect of temperature on phagocytosis and OB activity. Another critical step is the addition of bacteria to each sample. A 8:1 bacteria:phagocyte ratio produces optimal r.......
|12 x 75 mm tubes, with cap
|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
|Bottle top 0.2 µm cellulose acetate filter (500 ml capacity)
|Citric acid (anhydrous, cell culture tested, plant cell culture tested)
|Sodium citrate tribasic dihydrate
|Dihydroethidium (Hydroethidine) (HE)
|Dimethyl sulfoxide (DMSO) Anhydrous
|Staphylococcus aureus (Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)
|Staphylococcus aureus (Wood strain without protein A) BioParticles, unlabeled
|Trypan Blue Solution, 0.4%
|4.0 mL vacutainer containing 7.2 mg K2EDTA, spray-dried
|You will need 1 K2 EDTA blood collection tube per sample.
|4.0 mL vacutainer containing 68 USP units Lithium Heparin, spray-coated
|You will need 1 Lithium Heparin blood collection tube per sample.
|COULTER Ac·T 5diff CP
|i.e., hematology analyzer
|COULTER Ac·T 5diff Rinse
|COULTER Ac·T 5diff Fix
|COULTER Ac·T 5diff WBC Lyse
|COULTER Ac·T 5diff Hgb Lyse
|COULTER Ac·T 5diff Cal Calibrator
|COULTER Ac·T 5diff Control Plus
|AcT 5diff Diluent
|200 µL extended-length pipette tips
|Insulated ice pan
|For ice water bath.
|Open metal tube rack
|Fetal Bovine Serum
|i.e., automated cell lyse preparation workstation
|ImmunoPrep Reagent System
|i.e., RBC lyse/WBC fix reagent system
|Cytomics FC500 MCL Flow Cytometry System with CXP Software
|IsoFlow Sheath Fluid
|i.e., alignment and fluidics verification fluorospheres
|i.e., flow cytometry analysis software
|i.e., electronic spreadsheet program
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