Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

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

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....

Protocol

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

1. Gather, prepare, and organize the materials specified for the procedure (please refer to Table of Specific Materials/Equipment).
   1. Label 12 x 75 mm tubes.
      1. Label two tubes for each participant: one tube for the phagocytosis assay (label this tube with black ink and an "F" for fluorescein isothiocyanate [FITC]) and one tube for the OB activity assay (label this tube with red ink and an "H" for dihydro....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 K2EDTA 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