Name: measuring granulocyte and monocyte phagocytosis and oxidative burst activity in human blood
Uploaded: 2016-09-12T13:00:00
Description: Watch this Scientific Journal Video about Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood at JoVE.com

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

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
<ol>
	<li>Gather, prepare, and organize the materials specified for the procedure (please refer to Table of Specific Materials/Equipment).
	<ol>
		<li>Label 12 x 75 mm tubes.
		<ol>
			<li>Label two tubes for each participant: one tube for the phagocytosis assay (label this tube with black ink and an ...

<ol>
	<li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pistachios+on+performance+and+exercise-induced+inflammation,+oxidative+stress,+immune+dysfunction,+and+metabolite+shifts+in+cyclists:+a+randomized,+crossover+trial.">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><li>Knab, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+freeze-dried+juice+blend+powder+on+exercise-induced+inflammation,+oxidative+stress,+and+immune+function+in+cyclists.">Effects of a freeze-dried juice blend powder on exercise-induced inflammation, oxidative stress, and immune function in cyclists.</a> Appl Physiol Nutr Metab. 39, 381-385 (2014).</li><li>Konrad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+effect+of+ingesting+a+quercetin-based+supplement+on+exercise-induced+inflammation+and+immune+changes+in+runners.">The acute effect of ingesting a quercetin-based supplement on exercise-induced inflammation and immune changes in runners.</a> Int J Sport Nutr Exerc Metab. 21, 338-346 (2011).</li><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+and+inflammation+responses+to+a+3-day+period+of+intensified+running+versus+cycling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+supplementation+affects+blood+granulocyte+and+monocyte+trafficking+but+not+function+after+2.5+h+of+running.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+cell+response+to+acute+muscle+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+cytometry+reveals+three+distinct+subsets+of+activated+granulocytes+based+on+phagocytosis+and+oxidative+burst.">Image-based cytometry reveals three distinct subsets of activated granulocytes based on phagocytosis and oxidative burst.</a> Cytometry A. 83, 745-751 (2013).</li><li>Liao, Y., Liu, P., Guo, F., Zhang, Z. Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+burst+of+circulating+neutrophils+following+traumatic+brain+injury+in+human.">Oxidative burst of circulating neutrophils following traumatic brain injury in human.</a> PLoS One. 8, e68963 (2013).</li><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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+flow+cytometry+technique+to+evaluate+changes+in+granulocyte+function+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response+to+obesity+and+moderate+weight+loss.">Immune response to obesity and moderate weight loss.</a> Int J Obes Relat Metab Disord. 20, 353-360 (1996).</li><li>Nieman, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+function+in+female+elite+rowers+and+non-athletes.">Immune function in female elite rowers and non-athletes.</a> Br J Sports Med. 34, 181-187 (2000).</li><li>Ichii, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+sucrose+impairs+phagocytic+function+and+promotes+apoptosis+in+polymorphonuclear+leukocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probes+used+for+detection+of+reactive+oxygen+species.">Fluorescence probes used for detection of reactive oxygen species.</a> J Biochem Biophys Methods. 65, 45-80 (2005).</li><li>Gruger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+impact+of+linezolid+on+human+neutrophil+functions+in+vitro.">Negative impact of linezolid on human neutrophil functions in vitro.</a> Chemotherapy. 58, 206-211 (2012).</li><li>Dementhon, K., El-Kirat-Chatel, S., Noel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+in+vitro+model+for+the+multi-parametric+quantification+of+the+cellular+interactions+between+Candida+yeasts+and+phagocytes.">Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes.</a> PLoS One. 7, e32621 (2012).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1070">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">12 x 75 mm tubes, with cap</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">20170-579</td>
			<td style="width:425px;">You will need 2 tubes per sample (&#34;H&#34; and &#34;F&#34;) plus 3 tubes per batch (for assay controls; &#34;F&#34;, &#34;H&#34;, and &#34;Blood only&#34;).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PBS (10X), pH 7.4</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">70011-044</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bottle top 0.2 &#181;m cellulose acetate filter (500 ml capacity)</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:215px;">09-741-07&#160;</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glucose, powder</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">15023-021</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Citric acid (anhydrous, cell culture tested, plant cell culture tested)</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:215px;">C2404-100G</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Sodium citrate tribasic dihydrate</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:215px;">S4641-25G</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dihydroethidium (Hydroethidine) (HE)</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">D11347</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dimethyl sulfoxide (DMSO) Anhydrous</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">D12345</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:215px;">Staphylococcus aureus&#160;(Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">S2851</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Staphylococcus aureus&#160;(Wood strain without protein A) BioParticles, unlabeled</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">S-2859</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">15250-061</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">4.0 mL vacutainer containing 7.2 mg K2EDTA, spray-dried</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">BD367861</td>
			<td style="width:425px;">You will need 1 K2 EDTA blood collection tube per sample.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">4.0 mL vacutainer containing 68 USP units Lithium Heparin, spray-coated</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">BD367884</td>
			<td style="width:425px;">You will need 1 Lithium Heparin blood collection tube per sample.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff CP</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">6605705</td>
			<td style="width:425px;">i.e., hematology analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff Rinse</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547167</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff Fix</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547171</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff WBC Lyse</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547170</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER Ac&#183;T 5diff Hgb Lyse</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547168</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">COULTER Ac&#183;T 5diff Cal Calibrator</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">7547175</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">COULTER Ac&#183;T 5diff Control Plus</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">7547198</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">AcT 5diff Diluent</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547169</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">200 &#181;L extended-length pipette tips</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">37001-526</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Insulated ice pan</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">89198-980</td>
			<td style="width:425px;">For ice water bath.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Open metal tube rack</td>
			<td style="width:215px;">VWR</td>
			<td style="width:215px;">60916-702</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fetal Bovine Serum</td>
			<td style="width:215px;">Life Technologies</td>
			<td style="width:215px;">26140-111</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">TQ-Prep Workstation</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">6605429</td>
			<td style="width:425px;">i.e., automated cell lyse preparation workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">ImmunoPrep Reagent System</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">7546999</td>
			<td style="width:425px;">i.e., RBC lyse/WBC fix reagent system</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Cytomics FC500 MCL Flow Cytometry System with CXP Software</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">626553</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">IsoFlow Sheath Fluid</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8547008</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">COULTER CLENZ</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">8546929</td>
			<td style="width:425px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Flow-Check Fluorospheres&#160;</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:215px;">6605359</td>
			<td style="width:425px;">i.e., alignment and fluidics verification fluorospheres</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">FlowJo Software</td>
			<td style="width:215px;">FlowJo</td>
			<td style="width:215px;">FlowJo</td>
			<td style="width:425px;">i.e., flow cytometry analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Excel Software</td>
			<td style="width:215px;">Microsoft</td>
			<td style="width:215px;">Microsoft Excel</td>
			<td style="width:425px;">i.e., electronic spreadsheet program</td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood at JoVE.com

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

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.

measuring-granulocyte-monocyte-phagocytosis-oxidative-burst-activity

Research

JoVE Journal

Immunology and Infection

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...