The present study was funded in part by a Research Initiation Grant (RIG) from the University of North Texas to Dr. McFarlin. The authors did not receive direct funding for the completion of this study and report no conflict of interest.

NOTE: All blood collection procedures described in this method were conducted in accordance with the Declaration of Helsinki and approved by the UNT institutional review board (IRB) for human subjects. All subjects gave written consent for blood collection, which was used in the present method to ensure that they were apparently healthy, of normal body weight, and disease free.
1. Reagent Source &#38; Preparation
<ol>
	<li>Use CD66b-APC (clone#G10F5; DF=1:50) and CD45-APCeFluor780 (clone#2D1; DF=1:50) for this assay. 
	NOTE: Prior to the study, CD45 and CD66b antibodies were titered to determine the optimal dilution that clearly resolved granulocytes (CD45+/66b+) from other leukocytes (CD45+/66b-) 11. Upon addition of the diluted antibody for staining the final dilution in the tube was 875.</li>
	<li>Purchase and thaw stock S. aureus bioparticles labeled with pHrodo red dye. Suspend at a concentration of 1 mg of bioparticles per ml in sterile PBS. Aliquot dilute bioparticles and store frozen at -20 &#176;C until used in the assay.</li>
	<li>Dissolve dihydroethidium (DHE), which is converted to fluorescent ethidium bromide in the presence of oxygen-free radicals (i.e., the oxidative burst), in DMSO to a final concentration of 10 g/ml.</li>
	<li>Mix N-ethylmaleimide (used to prevent additional phagocytosis following the specified assay incubation duration) with sterile PBS in a 2 step dilution of 200 mg/ml and 17.5 mg/ml respectively. In the assay, use a final concentration of 15 mM. When thawing N-ethylmaleimide, use a 37 &#176;C heat block, as the N-ethylmaleimide does not remain in solution.</li>
	<li>After the final fixation step, use 7AAD to stain nuclear DNA. Prior to addition, dilute stock 7AAD 1:10 with sterile PBS.</li>
</ol>
2. Blood Sample Collection
<ol>
	<li>Ask subjects to arrive at the laboratory following an O/N fast (&#62;8hr) and abstention from physical activity (&#62;12 hr).</li>
	<li>After cleaning the skin with an alcohol prep pad, insert a sterile blood collection needle into a peripheral arm vein.</li>
	<li>Collect blood into evacuated tubes that are commercially filled with sodium heparin.</li>
	<li>After collection, apply an adhesive bandage to the subject&#8217;s arm.</li>
	<li>Invert blood tubes to mix 10 times and then place on a rocker until analysis.</li>
</ol>
3. Phagocytosis Assay Technique
<ol>
	<li>Thaw S.aureus bioparticles (RT), DHE (RT), and N-ethylmaleimide (37 &#176;C).</li>
	<li>Add 20 L of S.aureus bioparticles to 4 individual 1.2 ml tubes while working in the sterile hood.</li>
	<li>Add 40 L of DHE to each tube containing the bioparticles.</li>
	<li>Gently tap the tubes on the bench surface to collect the reagents in the bottom of the tube.</li>
	<li>Add 100 L of mixed whole blood to each tube that has been filled with bioparticles and DHE.</li>
	<li>Wipe the inside edge of the tubes with a cotton tipped applicator to remove any contaminating blood.</li>
	<li>Mix the blood and reagents with an electronic pipet set to mix the blood and reagents for three cycles after blood addition.</li>
	<li>Place assay tubes in an ice bucket and cover to protect from light.</li>
	<li>Incubate assay tubes for 10, 20, and 40 min. Start with the 40-min tube to ensure all assay tubes finish at the same time.</li>
	<li>Thaw N-ethylmaleimide in 37 &#176;C bead bath.</li>
	<li>Pipet 15 L of N-ethylmaleimide (described above in 1.4) into each assay tube, incubate for 30-min.</li>
	<li>Pipet 10 L of CD66b-APC and 10 L of CD45-APCeFluor780 diluted antibodies (described above in 1.1), incubate for 60 min.</li>
	<li>Pipet 750 L of WBC fix / RBC lyse solution into each assay tube, incubate for 60-min.</li>
	<li>Centrifuge (10 min at 400 x g) assay tubes to collect the cell pellet.</li>
	<li>Vacuum aspirate the WBC lyse / RBC fix solution, leaving a residual fluid volume (100 L) above the cell pellet.</li>
	<li>Pipet 10 L of diluted 7AAD solution (described above in 1.5) and 50 L of PBS into each assay tube.</li>
	<li>Add one drop of calibration beads (~25 L) to each assay tube, place caps on assay tubes, wrap tubes in foil, and place in a refrigerator.</li>
	<li>Load the sample tube into the image-based flow cytometer and collect a minimum of 3,000 granulocyte events using pre-defined parameters: Autosampler, blue (488 nm; 60 mW), red (640 nm; 100 nW), SSC (785 nm; 8.5 mW) lasers (Figure 1).</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52201/52201fig1highres.jpg" /> 
Figure 1. Acquisition Method &#38; Template. Samples were acquired on an image-based flow cytometer (A). During acquisition, dot plots of Bright Field Aspect Ratio vs. CD66b-APC were generated to separated granulocytes from spiked calibration beads using INSPIRE v. 100.2.292.0 software (B). A minimum of 5,000 granulocytes were acquired for each samples using an Autosampler, blue (488 nm; 60 mW), red (640 nm; 100 nW), SSC (785 nm; 8.5 mW) lasers. Please note that a number of histograms are present during acquisition to monitor laser settings and other aspects of data collect. All of these histograms are optional and only needed if the laboratory standard operating procedures requires them as quality control. <a href="https://www.jove.com/files/ftp_upload/52201/52201fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Sample Acquisition and Analysis
<ol>
	<li>Use the automated software compensation wizard of the IDEAS software to apply a compensation matrix to raw image files (RIF) and create compensate imaged files (CIF).</li>
	<li>Load individual CIF files in IDEAS software and generate the following plots to identify the granulocyte subsets:</li>
	<li>Establish initial gates to identify the cells that were considered to be in focus using a histogram for bright field gradient RMS (Figure 2A). Make this determination for each patient sample using the unstimulated control as a reference standard.</li>
	<li>Use secondary plots to separate singlet cells from debris and doublets using a dot plot of bright field aspect ratio (ratio of cell height vs. width) vs. bright field area (Figure 2B). Make this determination for each patient sample using the unstimulated control as a reference standard.</li>
	<li>Once a clean population of cells had been identified, establish a dot plot of CD45 vs. CD66b (Figure 2C) to positively identify granulocytes (CD45+/66b+). Create a daughter plot (Figure 3) of bright detail intensity for S. aureus (x-axis) vs. bright detail intensity for oxidative burst (DHE, y-axis) to identify subsets of activated granulocytes. Collect bright field images in Channel 1 and 9, bioparticles in Channel 2, DHE in Channel 4, 7AAD in Channel 5, CD66b in Channel 11, and CD45 in Channel 12.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52201/52201fig2highres.jpg" /> 
Figure 2.&#160;Analysis Template. This figure the series of plots that were generated to identify cells that were in focus (A), single cells (B), granulocytes (C). Additional plots were used to identify the three subsets of activated granulocytes vs. inactive granulocytes. All analysis of acquired image files was completed using IDEAS v.6 software. <a href="https://www.jove.com/files/ftp_upload/52201/52201fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Kong, 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+effect+of+alpha-fetoprotein+on+the+activation+and+phagocytosis+of+granulocytes+and+monocytes.">The effect of alpha-fetoprotein on the activation and phagocytosis of granulocytes and monocytes.</a> Hepatogastroenterology. 59, 2385-2388 (2012).</li><li>Prakash, P. S., Caldwell, C. C., Lentsch, A. B., Pritts, T. A., Robinson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microparticles+generated+during+sepsis+in+patients+with+critical+illness+are+neutrophil-derived+and+modulate+the+immune+response.">Human microparticles generated during sepsis in patients with critical illness are neutrophil-derived and modulate the immune response.</a> J. Trauma Acute Care Surg. 73, 401-406 (2012).</li><li>Salih, H. R., Husfeld, L., Adam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+cytofluorometric+measurement+of+phagocytosis,+burst+production+and+killing+of+human+phagocytes+using+Candida+albicans+and+Staphylococcus+aureus+as+target+organisms.">Simultaneous cytofluorometric measurement of phagocytosis, burst production and killing of human phagocytes using Candida albicans and Staphylococcus aureus as target organisms.</a> Clin. Microbiol. Infect. 6, 251-258 (2000).</li><li>Tsuji, S., Iharada, A., Taniuchi, S., Hasui, M., Kaneko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+production+of+nitric+oxide+by+phagocytic+stimulated+neutrophils+in+patients+with+chronic+granulomatous+disease.">Increased production of nitric oxide by phagocytic stimulated neutrophils in patients with chronic granulomatous disease.</a> J. Pediatr. Hematol. Oncol. 34, 500-502 (2012).</li><li>Ortyn, W. E., 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=Sensitivity+measurement+and+compensation+in+spectral+imaging.">Sensitivity measurement and compensation in spectral imaging.</a> Cytometry A. 69, 852-862 (2006).</li><li>McFarlin, B. K., 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=A+one-year+school-based+diet/exercise+intervention+improves+non-traditional+disease+biomarkers+in+Mexican-American+children.">A one-year school-based diet/exercise intervention improves non-traditional disease biomarkers in Mexican-American children.</a> Matern. Child Nutr. 9, 524-532 (2013).</li><li>McFarlin, B. K., Johnson, C. A., Moreno, J. P., Foreyt, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mexican-American+Children+have+differential+elevation+of+Metabolic+Biomarkers+that+is+proportional+to+Obesity+Status.">Mexican-American Children have differential elevation of Metabolic Biomarkers that is proportional to Obesity Status.</a> J. Pediatr. Gastroenterol. Nutr. , (2013).</li><li>Strohacker, K., 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=Moderate-intensity,+premeal+cycling+blunts+postprandial+increases+in+monocyte+cell+surface+CD18+and+CD11a+and+endothelial+microparticles+following+a+high-fat+meal+in+young+adults.">Moderate-intensity, premeal cycling blunts postprandial increases in monocyte cell surface CD18 and CD11a and endothelial microparticles following a high-fat meal in young adults.</a> Appl. Physiol. Nutr. Metab. 37, 530-539 (2012).</li><li>Carpenter, K. C., Breslin, W. L., Davidson, T., Adams, A., McFarlin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baker's+yeast+beta-glucan+supplementation+increases+monocytes+and+cytokines+post-exercise:+implications+for+infection+risk.">Baker's yeast beta-glucan supplementation increases monocytes and cytokines post-exercise: implications for infection risk.</a> Br. J. Nutr. , 1-9 (2012).</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 (1002).</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>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>Ploppa, A., George, T. C., Unertl, K. E., Nohe, B., Durieux, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageStream+cytometry+extends+the+analysis+of+phagocytosis+and+oxidative.">ImageStream cytometry extends the analysis of phagocytosis and oxidative.</a> Scand. J. Clin. Lab. Invest. 71, 362-369 (2011).</li><li>Van Amersfoort, E. S., Van Strijp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+flow+cytometric+fluorescence+quenching+assay+of+phagocytosis+of+sensitized+sheep+erythrocytes+by+polymorphonuclear+leukocytes.">Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes.</a> Cytometry. 17, 294-301 (1994).</li><li>Gullstrand, B., 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=Combination+of+autoantibodies+against+different+histone+proteins+influences+complement-dependent+phagocytosis+of+necrotic+cell+material+by+polymorphonuclear+leukocytes+in+systemic+lupus+erythematosus.">Combination of autoantibodies against different histone proteins influences complement-dependent phagocytosis of necrotic cell material by polymorphonuclear leukocytes in systemic lupus erythematosus.</a> J. Rheumatol. 39, 1619-1627 (2012).</li><li>Fierro, M. 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=Functional+and+phenotypical+impairment+of+polymorphonuclear+cells+in+atopic+dermatitis:+an+additional+cause+for+the+known+susceptibility+to+infections.">Functional and phenotypical impairment of polymorphonuclear cells in atopic dermatitis: an additional cause for the known susceptibility to infections.</a> Dermatology. 224, 323-330 (2012).</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>McFarlin, B. K., Venable, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Low+Concentration+Human+Serum+Cytokines+using+a+Millipore+High-Sensitivity+Milliplex+Assay.">Measurement of Low Concentration Human Serum Cytokines using a Millipore High-Sensitivity Milliplex Assay.</a> J. Vis. Exp. , (2014).</li><li>McFarlin, B. K., Carpenter, K. C., Davidson, T., McFarlin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baker's+Yeast+Beta+Glucan+Supplementation+Increases+Salivary+IgA+and+Decreases+Cold/Flu+Symptomatic+Days+After+Intense+Exercise.">Baker's Yeast Beta Glucan Supplementation Increases Salivary IgA and Decreases Cold/Flu Symptomatic Days After Intense Exercise.</a> J. Diet. Suppl. 10, 171-183 (2013).</li><li>McFarlin, B. K., Carpenter, K. C., Strohacker, K., Breslin, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+blood+monocytes+and+adipose+tissue+macrophages+in+a+mouse+model+diet-induced+weight+gain.">Comparison of blood monocytes and adipose tissue macrophages in a mouse model diet-induced weight gain.</a> Comp. Med. 62, 462-465 (2012).</li></ol>

The authors have nothing to disclose.

The present method represents a refinement of existing methods for the assessment of granulocyte function using flow cytometry 1,3,4,12-14. The critical steps of this assay tend to be related to proper mixing of the blood sample with the bioparticles and DHE. Incomplete mixing will result in inaccurate results. While complete mixing is critical, the mixing method should be gentle in nature. It is suggested that mixing be accomplished using an electronic pipet with a mixing function rather than a vortex mixer. Another critical step in the assay is to always ensure there is no blood contaminating the upper half of the assay tube. This residual blood can be removed using a sterile cotton-tipped applicator. Complete removal is important because failure to do so may contaminate the final assay preparation with un-lysed red blood cells.
Prior to using this method appropriate compensation controls should be added to control for spectral overlap amongst the reagents used to identify the various aspects of granulocyte function. For this method, compensation controls involve collect blood samples that have been suggested the 40 min assay incubation and then labeling with a single marker (i.e.,&#160;E. coli, DHE, etc.). After labeling, single positive events are collected and a compensation matrix is generated using an automated wizard in the IDEAS analysis software. It is critical that if this assay is used, appropriate compensation controls are completed to ensure proper assay performance.
Analysis is accomplished using the feature finder and co-localization wizards to identify that bright detail intensity is the best variable to separate the populations and also identify how much overlap exists between oxidative burst and phagocytosis signals. Specifically, the use of image-based cytometry provided the ability to segregate activated granulocytes into three subsets. This subset breakdown was determined using bright detail intensity of phagocytosis vs. oxidative burst. In addition to examining these singular cell functions, cells that exhibited both events at the same time in the same anatomical location (co-localization) were identified. Granulocytes that fell into the &#8220;high-active&#8221; subset were the only phenotype that demonstrated consistent co-localization between phagocytosis and oxidative burst. This identification of activated granulocyte subsets is the biggest area where troubleshooting is needed. It is very important for a new user to take time to understand the sample process workflow in IDEAS and to understand the mechanics of gating the cell populations using the imaging component. Other modifications that a user may seek to make include the selection of alternate or additional assay incubation times. The current method suggests the use of durations of 10-40 min; however, depending on the experimental model where this method is to be used, it may be necessary to select longer assay durations. Such a modification would need to be evaluated on an individual basis.
Further it was determined that the duration of the assay incubation has a significant effect on the appearance of the three activated subsets. The approach described in this report represents an extension of what information could be previously obtained regarding granulocyte function 3,4,13,15. Other laboratories have demonstrated the importance of assessing changes in granulocyte function as part of a comprehensive assessment of immunity and disease 15-17. Despite the potential of this assay it is not without limitations. One of the major limitations is cost and time demand associated with high sample throughput processing. When a study design requires a large number of samples in a given day, these can be difficult to process. Processing is streamlined by the use of electronic pipets and dispensers, but these tend to be expensive and are not necessarily available in every laboratory.
Our area of research focuses on a study of how exercise and dietary habits influence immune system health and function 6,8-10,18-20. Such objectives have significant practical implications for a variety of areas of human health. Beyond the study of exercise and nutritional effects the phagocytosis method demonstrated in this manuscript could be useful in other areas of clinical immunology where the monitoring of phagocytosis function is critical to treatment outcomes. The present assay is the first of many that immunological assays with the potential to be re-invented by taking advantages of the unique imaging information that can be can from an image-based flow cytometer.

Granulocytes represent one component of the body&#8217;s innate immune system, which provides the first line of defense against invading antigens. Previous methods for assessing granulocyte function focused on phagocytosis capacity or oxidative burst using separate methods, making it difficult to ascertain collectively how granulocytes have changed 1-4. Advances in the field of flow cytometry have resulted in the production of benchtop instruments capable of high-resolution, multi-color imaging of cells in a high throughput manner 5. The ability to combine imaging with traditional flow cytometry represents an advancement that provides the technological platform needed to innovate within existing flow cytometry methods and extract new information about the immune system.
Over the past ten years, our laboratory, among others, has been keenly focused on the effect of various nutritional and exercise treatments patterns on innate immune function 6-9. The method demonstrated in this manuscript has practical implications within the field of clinical immunology. The present method leverages the power of image-based flow cytometry to simultaneously measure phagocytosis of bacterial particles and oxidative burst. Using this approach, one is able to separate activated granulocytes using variables provided by the image-based part of the analysis. These subsets were only identifiable after assessing the cellular images on the individual granulocytes. Further assay incubation time affected the transition between the three activation subsets 10. Thus, it is plausible that use of multiple incubation times may allow a method to test the change in granulocyte function following a specific experimental treatment. The purpose of this manuscript was to demonstrate a method of assessing granulocyte function by using image-based flow cytometry to simultaneously measure phagocytosis with oxidative burst.

<table><tbody><tr><td>Vacutainers</td><td>BD Life Sciences</td><td></td><td>used for blood collection</td></tr><tr><td>WBC Fixative / RBC Lysis solution</td><td>eBioscience</td><td>00-5333-57</td><td></td></tr><tr><td>CD66b-APC</td><td>eBioscience</td><td>clone G10F5</td><td></td></tr><tr><td>CD45-APCeFluor780</td><td>eBioscience</td><td>clone 2D1</td><td></td></tr><tr><td>&lt;i&gt;S. aureus&lt;/i&gt; bioparticles</td><td>Life Technologies</td><td>A10010</td><td></td></tr><tr><td>dihydroethidium</td><td>Sigma-Aldrich</td><td>D7008</td><td></td></tr><tr><td>N-ethylmalemide</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>7AAD</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>Hematology Analyzer</td><td>Mindray</td><td>BC-3200</td><td></td></tr><tr><td>96-channel pipet</td><td>Integra Biosciences</td><td>ViaFlo</td><td></td></tr><tr><td>Bead Bath Incubator</td><td>LabArmour</td><td>BeadBath</td><td></td></tr><tr><td>Imaging Flow Cytometer</td><td>EMD Millipore</td><td>Amnis FlowSight</td><td></td></tr><tr><td>INSPIRE Software</td><td>EMD Millipore</td><td>Amnis INSPIRE</td><td></td></tr><tr><td>IDEAS Software</td><td>EMD Millipore</td><td>Amnis IDEAS</td><td></td></tr><tr><td>X-Pierce Piercable Plate Sealer</td><td>Excel Scientific, Inc.</td><td>X-Pierce</td><td></td></tr><tr><td>Dell Precision Workstation</td><td>Dell Computers</td><td>Various</td><td>Used for IDEAS analysis</td></tr></tbody></table>

image-based flow cytometry technique to evaluate changes in granulocyte function in vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Using image-based flow cytometry allowed us to separate a homogenous population of activated granulocytes into three different activation subsets (Figure 3). In this method, the most effective way to resolve the three activation subsets is by plotting bright detail intensity for phagocytosis (S. aureus) vs. oxidative burst (DHE) (Figure 3; Figure 4). Also, use of the co-localization wizard in IDEAS software will allow for the quantification of the presence of simultaneous phagocytosis and oxidative burst, which is a hallmark sign of highly activated granulocytes.
<img alt="Figure 3" src="/files/ftp_upload/52201/52201fig3highres.jpg" /> 
Figure 3.&#160;Identification of Activated Granulocyte Subsets. After the identification of granulocytes using cell-surface markers (CD45+/66b+), a daughter plot was generated with bright detail intensity for phagocytosis vs. bright detail intensity for oxidative burst. Using this approach, percentage of inactive (purple), low-active (red, A), moderate-active (blue, B), and high-active (yellow, C) granulocytes was determined. This gating technique was also used to evaluate the effect of assay incubation time on the relative abundance of the various activated granulocyte subsets. The highest percentage of &#8220;high-active&#8221; granulocytes was present following the 40 min incubation. All analysis of acquired image files was completed using IDEAS v.6 software. <a href="https://www.jove.com/files/ftp_upload/52201/52201fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In addition to demonstrating the presence of three distinct subsets of activated granulocytes, it was demonstrated that the assay incubation time influenced the relative abundance of each activated granulocytes subset. Specifically, the 40 min incubation resulted in the greatest percentage of &#8220;high-active&#8221; granulocytes. By including at least three assay incubation durations it may be possible to determine how a given clinical treatment alters the temporal activation status of granulocytes. This is the first published method using image-based flow cytometry to identify different activated granulocyte subsets as a function of simultaneous measurements of phagocytosis and oxidative burst.
<img alt="Figure 4" src="/files/ftp_upload/52201/52201fig4highres.jpg" /> 
Figure 4.&#160;Representative Images of Cellular Markers. An image gallery of cells classified as high-active (A), moderate-active (B), and low-active (C) is presented in this figure. S.aureus bioparticles are in Ch03, oxidative burst is in Ch04, 7AAD for the nucleus is in Ch05, side scatter is in Ch06, CD66b is in Ch11, and CD45 is in Ch12. Also, a co-localized merge image of phagocytosis (Ch03) vs. oxidative burst (Ch04) is displayed. Areas of yellow in the merge image denote that phagocytosis and oxidative burst are occurring in the same anatomical space at the same time. <a href="https://www.jove.com/files/ftp_upload/52201/52201fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro at JoVE.com

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

image-based-flow-cytometry-technique-to-evaluate-changes-granulocyte

Research

JoVE Journal

Immunology and Infection

12.6K Views.  University of North Texas. This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Video: Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

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.

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 ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

Neuroscience

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Biology

Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry

Source: Do&#241;a, I. et al., <a href="https://app.jove.com/v/62600">Basophil Activation Test for Allergy Diagnosis.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the test to evaluate basophil activation in the presence of a test allergen using flow cytometry.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sample preparation

	Collect ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry

Source: Murinello, S. et al., <a href="https://app.jove.com/v/54677">Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay.</a> J. Vis. Exp.&#160;(2016)
This video demonstrates flow cytometry for measuring microglial uptake of fluorescent bioparticles after intravitreal delivery in a mouse eye. Flow cytometry detects live cells expressing CD11b and distinguishes phagocytic microglia cells exhibiting green fluorescence from other myeloid cells, including Ly6C-expressing monocytes and Ly6G-expressing neutrophils.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Summary

Explore More Videos

Chapters in this video

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

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

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Flow Cytometric Characterization of Murine B Cell Development

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry

Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry