Name: Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood (Video) | JoVE Journal
Uploaded: 2016-09-12T13:00:00.000+00:00
Duration: 11 min 29 s
Description: Learn to measure granulocyte and monocyte phagocytosis and oxidative burst activity in human blood using flow cytometry. This adaptable assay supports immunology research on exercise and dietary interventions.

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5 minutes

The authors would like to acknowledge Zack Shue, Casey John, and Lynn Cialdella-Kam for their assistance during the optimization of this procedure.

<ol><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+pistachios+on+performance+and+exercise-induced+inflammation%2C+oxidative+stress%2C+immune+dysfunction%2C+and+metabolite+shifts+in+cyclists%3A+a+randomized%2C+crossover+trial%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Influence of pistachios on performance and exercise-induced inflammation, oxidative stress, immune dysfunction, and metabolite shifts in cyclists: a randomized, crossover trial.</a> PLoS One. 9, e113725(2014).</li>
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<li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+and+inflammation+responses+to+a+3-day+period+of+intensified+running+versus+cycling%5BTitle%5D)+AND+%22Brain+Behav+Immun%22%5BJournal%5D)">Immune and inflammation responses to a 3-day period of intensified running versus cycling.</a> Brain Behav Immun. 39, 180-185 (2014).</li>
<li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Carbohydrate+supplementation+affects+blood+granulocyte+and+monocyte+trafficking+but+not+function+after+2.5+h+of+running%5BTitle%5D)+AND+%22Am+J+Clin+Nutr%22%5BJournal%5D)">Carbohydrate supplementation affects blood granulocyte and monocyte trafficking but not function after 2.5 h of running.</a> Am J Clin Nutr. 66, 153-159 (1997).</li>
<li>Tidball, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+cell+response+to+acute+muscle+injury%5BTitle%5D)+AND+%22Med+Sci+Sports+Exerc%22%5BJournal%5D)">Inflammatory cell response to acute muscle injury.</a> Med Sci Sports Exerc. 27, 1022-1032 (1995).</li>
<li>McFarlin, B. K., Williams, R. R., Venable, A. S., Dwyer, K. C., Haviland, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-based+cytometry+reveals+three+distinct+subsets+of+activated+granulocytes+based+on+phagocytosis+and+oxidative+burst%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Image-based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst.</a> Cytometry A. 83, 745-751 (2013).</li>
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<li>McFarlin, B. K., Venable, A. S., Prado, E. A., Henning, A. L., Williams, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-based+flow+cytometry+technique+to+evaluate+changes+in+granulocyte+function+in+vitro%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Image-based flow cytometry technique to evaluate changes in granulocyte function in vitro.</a> J Vis Exp. , (2014).</li>
<li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+response+to+obesity+and+moderate+weight+loss%5BTitle%5D)+AND+%22Int+J+Obes+Relat+Metab+Disord%22%5BJournal%5D)">Immune response to obesity and moderate weight loss.</a> Int J Obes Relat Metab Disord. 20, 353-360 (1996).</li>
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<li>Ichii, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron+sucrose+impairs+phagocytic+function+and+promotes+apoptosis+in+polymorphonuclear+leukocytes%5BTitle%5D)+AND+%22Am+J+Nephrol%22%5BJournal%5D)">Iron sucrose impairs phagocytic function and promotes apoptosis in polymorphonuclear leukocytes.</a> Am J Nephrol. 36, 50-57 (2012).</li>
<li>Gomes, A., Fernandes, E., Lima, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+probes+used+for+detection+of+reactive+oxygen+species%5BTitle%5D)+AND+%22J+Biochem+Biophys+Methods%22%5BJournal%5D)">Fluorescence probes used for detection of reactive oxygen species.</a> J Biochem Biophys Methods. 65, 45-80 (2005).</li>
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The authors have nothing to disclose.

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...

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 activity are the most common measures of granulocyte and monocyte function,7 and are preferred as indicators of innate immune cell function because of their central role in both the infection process and the repair process.
The assay described in this manuscript utilizes a basic flow cytometer to provide a quantitative determination of granulocyte and monocyte phagocytosis and OB activity in whole blood. The use of whole blood in this technique is advantageous because it allows the assay to be conducted without a time-consuming purification procedure. This assay can easily be modified to accommodate a variety of research designs and lab capabilities. For example, Liao et al. utilized a similar technique to show that neutrophil OB activity is increased and neutrophil phagocytosis is decreased following traumatic brain injury.8 In another example, McFarlin et al. described an elegant modification of this technique that uses allophycocyanin (APC)-conjugated CD66b and high-performance tandem APC-conjugated CD45 labeling with image-based flow cytometry to classify granulocytes in terms of their phagocytosis and OB activities.7,9
Our research group has used various adaptations of this technique as an indicator of innate immune function in overweight, obese, and athletic populations for almost 20 years.10,11 The text below will provide the reader with step-by-step instructions for performing this assay and highlight areas that the reader may adapt to meet the needs of their research.

<table><tbody><tr><td>12 x 75 mm tubes, with cap</td><td>VWR</td><td>20170-579</td><td>You will need 2 tubes per sample (&quot;H&quot; and &quot;F&quot;) plus 3 tubes per batch (for assay controls; &quot;F&quot;, &quot;H&quot;, and &quot;Blood only&quot;).</td></tr><tr><td>PBS (10x), pH 7.4</td><td>Life Technologies</td><td>70011-044</td><td></td></tr><tr><td>Bottle top 0.2 &amp;micro;m cellulose acetate filter (500 ml capacity)</td><td>Fisher Scientific</td><td>09-741-07&amp;nbsp;</td><td></td></tr><tr><td>Glucose, powder</td><td>Life Technologies</td><td>15023-021</td><td></td></tr><tr><td>Citric acid (anhydrous, cell culture tested, plant cell culture tested)</td><td>Sigma-Aldrich</td><td>C2404-100G</td><td></td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma-Aldrich</td><td>S4641-25G</td><td></td></tr><tr><td>Dihydroethidium (Hydroethidine) (HE)</td><td>Life Technologies</td><td>D11347</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO) Anhydrous</td><td>Life Technologies</td><td>D12345</td><td></td></tr><tr><td>Staphylococcus aureus&amp;nbsp;(Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)</td><td>Life Technologies</td><td>S2851</td><td></td></tr><tr><td>Staphylococcus aureus&amp;nbsp;(Wood strain without protein A) BioParticles, unlabeled</td><td>Life Technologies</td><td>S-2859</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>4.0 ml vacutainer containing 7.2 mg K&lt;sub&gt;2&lt;/sub&gt;EDTA, spray-dried</td><td>VWR</td><td>BD367861</td><td>You will need 1 K&lt;sub&gt;2&lt;/sub&gt;EDTA blood collection tube per sample.</td></tr><tr><td>4.0 ml vacutainer containing 68 USP units Lithium Heparin, spray-coated</td><td>VWR</td><td>BD367884</td><td>You will need 1 Lithium Heparin blood collection tube per sample.</td></tr><tr><td>COULTER Ac&amp;middot;T 5diff CP</td><td>Beckman Coulter</td><td>6605705</td><td>&lt;em&gt;i.e.&lt;/em&gt;, hematology analyzer</td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Rinse</td><td>Beckman Coulter</td><td>8547167</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Fix</td><td>Beckman Coulter</td><td>8547171</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff WBC Lyse</td><td>Beckman Coulter</td><td>8547170</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Hgb Lyse</td><td>Beckman Coulter</td><td>8547168</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Cal Calibrator</td><td>Beckman Coulter</td><td>7547175</td><td></td></tr><tr><td>COULTER Ac&amp;middot;T 5diff Control Plus</td><td>Beckman Coulter</td><td>7547198</td><td></td></tr><tr><td>AcT 5diff Diluent</td><td>Beckman Coulter</td><td>8547169</td><td></td></tr><tr><td>200 &amp;micro;l extended-length pipette tips</td><td>VWR</td><td>37001-526</td><td></td></tr><tr><td>Insulated ice pan</td><td>VWR</td><td>89198-980</td><td>For ice water bath.</td></tr><tr><td>Open metal tube rack</td><td>VWR</td><td>60916-702</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>TQ-Prep Workstation</td><td>Beckman Coulter</td><td>6605429</td><td>&lt;em&gt;i.e.&lt;/em&gt;, automated cell lyse preparation workstation</td></tr><tr><td>ImmunoPrep Reagent System</td><td>Beckman Coulter</td><td>7546999</td><td>&lt;em&gt;i.e.&lt;/em&gt;, RBC lyse/WBC fix reagent system</td></tr><tr><td>Cytomics FC500 MCL Flow Cytometry System with CXP Software</td><td>Beckman Coulter</td><td>626553</td><td></td></tr><tr><td>IsoFlow Sheath Fluid</td><td>Beckman Coulter</td><td>8547008</td><td></td></tr><tr><td>COULTER CLENZ</td><td>Beckman Coulter</td><td>8546929</td><td></td></tr><tr><td>Flow-Check Fluorospheres&amp;nbsp;</td><td>Beckman Coulter</td><td>6605359</td><td>&lt;em&gt;i.e.&lt;/em&gt;, alignment and fluidics verification fluorospheres</td></tr><tr><td>FlowJo Software</td><td>FlowJo</td><td>FlowJo</td><td>&lt;em&gt;i.e.&lt;/em&gt;, flow cytometry analysis software</td></tr><tr><td>Excel Software</td><td>Microsoft</td><td>Microsoft Excel</td><td>&lt;em&gt;i.e.&lt;/em&gt;, electronic spreadsheet program</td></tr></tbody></table>

measuring granulocyte and monocyte phagocytosis and oxidative burst activity in human blood

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

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.

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...

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 overall goal of this assay is to measure granulocyte and monocyte phagocytosis and oxidative burst activity in whole blood. This method can help answer key questions in the field of immunology, such as how does a particular dietary intervention affect the immune response to exercise. The main advantage of this technique is that it is a simple, straightforward assay that can be easily modified to account for the available laboratory resources and research interests.We have been using this measure of innate immune function for the past two decades in many of our exercise trials, and we'll share some of these data later in the video. Before beginning the procedure, use a hematology analyzer to perform a complete blood count with white blood cell differential analysis on the blood samples according to the manufacturer's instructions. Make a note of the white blood cell count in cells per milliliter and the percent neutrophil and monocyte values for the samples.Then, for each experimental and control tube, use an extended length pipette tip to transfer 100 microliters of each whole blood sample from the lithium heparin blood collection tube to the bottom of an appropriately labeled 12 by 75 millimeter tube. When all of the blood has been transferred, add 10 microliters of the HE working solution to the tubes labeled with red ink and an H, including the HE control tube. Uncap the tubes.Vortex the samples briefly, and transfer the tubes to a 37-degree Celsius water bath in an open metal rack with gentle shaking every five minutes. After 15 minutes, quickly transfer the rack and tubes to an ice water bath for 12 minutes with gentle shaking every five minutes. At the end of the incubation, add the appropriate volume of unlabeled S.aureus working solution to the tubes labeled with the red ink and an H, including the HE control tube.Vortex the tubes briefly with the caps. Then add the appropriate volume of FITC-labeled S.aureus working solution to the tubes labeled with black ink and an F, including the FITC control tube, and briefly vortex the samples. Next, incubate all of the tubes in the 37-degree Celsius water bath for 20 minutes with shaking every five minutes followed by a one-minute cooldown in the ice water.After placing the samples at room temperature, use a repeater pipette to add 100 microliters of ice-cold quench solution to all of the tubes and vortex the samples. Return the samples to the ice bath. After one minute, use the repeater pipette to add one millimeter of ice-cold PBS to each of the tubes.Then vortex the tubes and add an additional two milliliters of PBS to the samples. Now centrifuge the cells and aspirate the supernatant. Using the repeater pipette, add 50 microliters of ice-cold FBS to the tubes.After vortexing, transfer all the tubes to a carousel. Use an automated cell lysis preparation workstation to lyse the red blood cells and fix the white blood cells according to the manufacturer's instructions. To analyze the assay results by flow cytometry, open the flow cytometry system acquisition software and create a new protocol.Choose FITC for the FL1 parameter for the phagocytosis assay and HE for the FL2 parameter for the oxidative burst activity assay. Create a forward versus side scatter dot plot, an FL1 histogram, and an FL2 histogram. Then create polygonal regions for the white blood cell, granulocyte and monocyte populations on the forward versus side scatter plot and create linear regions on the histograms.Now run the blood-only negative control sample followed by the HE and FITC positive control samples, reviewing the histograms and adjusting the photomultiplier tube voltages of the FITC and PE channels as necessary. Then start the data acquisition for the control samples. After a 15-minute equilibration period, load the experimental samples into the carousel according to the order on the work list and being the study sample analysis.To assess the phagocytic activity of the samples, first consolidate all of the F-labeled samples and control list mode data files into a single folder and drag the files into the sample space in the flow cytometry analysis software. Double-click on a control sample and use the polygon gate selector to set a gate for the total white blood cell population, taking care to include all of the cells at the edges of the chart. Label this gate WBC.Double-click on the WBC gate and use the elliptical gate selector to set the lymphocyte gate. Label the gate accordingly and drag the gate data percent to the side so that the cells are easily viewed. Then use the polygon gate selector to set the granulocyte gate and monocyte gate.Label each gate accordingly and drag the gate data percent to the side. Double-click on the FITC control sample followed by the granulocyte gate and use the drop-down menus to set the X-axis to FL1 and the Y-axis to side scatter, or SS.Use the square gate selector to set a phagocytosis negative gate and a phagocytosis positive gate. Highlight the phagocytosis positive gate of the granulocyte population.Then click Workspace and Add Statistic. Highlight Geometric Mean and FL1 to generate the fluorescence intensity of the phagocytosis positive granulocytes and click Add. Then choose the frequency of parent statistic and click Add to generate the percentage of the granulocytes that are phagocytosis positive.To apply these settings to all of the study samples, highlight all of the gates and statistics created in the FITC control sample, and drag them into the All Samples area in the group panel. Save the work as a WSP file in the same folder that contains the list mode data folder. Now click on the first experimental sample in the data set and confirm that the white blood cell gate appropriately fits the cell distribution on each of the samples, adjusting the edges of the gate as necessary.Now click on the white blood cell gate in the first experimental sample and confirm that the granulocyte, monocyte, and lymphocyte gates fit the cell distribution on each of the samples, adjusting the gates as necessary. After saving the file, click on the table editor icon, and drag each statistic of interest into the table. Click Create Table to display the data in a spreadsheet format and save the work as an XLSX file.Then repeat the analysis for the H-labeled oxidative burst activity samples and control list mode data files as just demonstrated. As demonstrated in this representative experiment, the granulocyte-mediated phagocytosis of S.aureus is increased immediately and 1.5 hours after a 75-kilometer cycling bout, returning to normal by the next morning. Granulocyte oxidative burst activity is decreased for at least 21 hours after this level of activity.Further, there is a modest correlation between granulocyte phagocytosis and serum C-reactive protein levels, as observed in this analysis of 106 overweight or obese women, indicating that high granulocyte and monocyte phagocytosis in resting subjects may be indicative of systemic inflammation. Once mastered, this technique can be completed for approximately 30 samples in seven hours if performed properly. While attempting this procedure, it's important to remember to completely cool the samples before adding the bacteria to help minimize any unexplained between-sample variability.Following this procedure, other methods, like cytokine quantification, can be performed to determine the effect of your intervention on the inflammatory response. After its development, this technique paved the way for researchers in the field of immunology to explore the effects of a variety of lifestyle changes and innate immune function in humans. After watching this video, you should have a good understanding of how to measure granulocyte and monocyte phagocytosis and oxidative burst activity with a flow cytometer.Don't forget that working with human blood can be extremely hazardous and that precautions, such as wearing the appropriate PPE, should always be taken when performing this procedure.

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Research

JoVE Journal

Immunology and Infection

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Learn to measure granulocyte and monocyte phagocytosis and oxidative burst activity in human blood using flow cytometry. This adaptable assay supports immunology research on exercise and dietary interventions.

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

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