Name: a simple flow cytometric method to measure glucose uptake and glucose transporter expression for monocyte subpopulations in whole blood
Uploaded: 2016-08-12T14:00:00
Description: Watch this Scientific Journal Video about A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood at JoVE.com

This research was funded by the Australian Centre for HIV and Hepatitis Virology Research (ACH2) and a 2010 developmental grant (CNIHR) from the University of Washington Center for AIDS Research (CFAR), an NIH funded program under award number AI027757 which is supported by the following NIH Institutes and Centers (NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, NIA). C.S.P is a recipient of the CNIHR and ACH2 grant. SMC is a recipient of a National Health and Medical Research Council of Australia (NHMRC) Principal Research Fellowship. The authors gratefully acknowledge the contribution to this work of the Victorian Operational Infrastructure Support Program received by the Burnet Institute. We acknowledge the assistance of Geza Paukovic and Eva Orlowski-Oliver from the AMREP Flow Cytometry Core Facility for flow cytometry training and technical advice. We thank Angus Morgan for media coaching and organization of the video shoot.&#160; Our gratitude to Jesse Masson and Jehad Abdulaziz K. Alzahrani for lab assistance during the video shoot.&#160;We thank the efforts of Dr David Simar at the School of Medical Sciences, UNSW, Australia who offered critical methodological advice. C.S.P would like to thank <a href="http://www.nice-consultants.com/">www.nice-consultants.com</a> for graphic consultations.
AUTHORS&#39; CONTRIBUTION:
C.S.P conceived the project, designed and conducted experiments, analyzed and interpreted data, and wrote the manuscript. J.J.A interpreted data and wrote the manuscript. T.R.B wrote the manuscript. J.M.M interpreted data, made critical intellectual suggestions, and reviewed the manuscript. S.M.C interpreted data, made critical intellectual suggestions and reviewed the manuscript.

NOTE: HIV-infected and HIV-uninfected subjects were recruited from the Infectious Diseases Unit at The Alfred Hospital in Melbourne, VIC, Australia, and from the local community, respectively. Informed consent was obtained from all participants, and the research was approved by The Alfred Hospital Research and Ethics Committee.
1. Glut1 Cell Surface Detection on Monocytes and Monocyte Subpopulations
<ol>
	<li>Collect blood in citrate ACD-B anticoagulant tubes and begin the experiments in a b...

<ol>
	<li>Shi, C., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+recruitment+during+infection+and+inflammation.">Monocyte recruitment during infection and inflammation.</a> Nat Rev Immunol. 11, 762-774 (2011).</li><li>Woollard, K. J., Geissmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+in+atherosclerosis:+subsets+and+functions.">Monocytes in atherosclerosis: subsets and functions.</a> Nat Rev Cardiol. 7, 77-86 (2010).</li><li>Richards, D. M., Hettinger, J., Feuerer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+macrophages+in+cancer:+development+and+functions.">Monocytes and macrophages in cancer: development and functions.</a> Cancer Microenviron. 6, 179-191 (2013).</li><li>Anzinger, J. J., Butterfield, T. R., Angelovich, T. A., Crowe, S. M., Palmer, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+as+regulators+of+inflammation+and+HIV-related+comorbidities+during+cART.">Monocytes as regulators of inflammation and HIV-related comorbidities during cART.</a> J Immunol Res. 2014, 569819 (2014).</li><li>Palmer, C., Cherry, C. L., Sada-Ovalle, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+Metabolism+in+T+Cells+and+Monocytes:+New+Perspectives+in+HIV+Pathogenesis.">Glucose Metabolism in T Cells and Monocytes: New Perspectives in HIV Pathogenesis.</a> EBioMedicine. , (2016).</li><li>Cheng, S. 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=mTOR-+and+HIF-1alpha-mediated+aerobic+glycolysis+as+metabolic+basis+for+trained+immunity.">mTOR- and HIF-1alpha-mediated aerobic glycolysis as metabolic basis for trained immunity.</a> Science. 345, 1250684 (2014).</li><li>Maratou, 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=Glucose+transporter+expression+on+the+plasma+membrane+of+resting+and+activated+white+blood+cells.">Glucose transporter expression on the plasma membrane of resting and activated white blood cells.</a> Eur J Clin Invest. 37, 282-290 (2007).</li><li>Freemerman, A. J., 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=Metabolic+reprogramming+of+macrophages:+glucose+transporter+1+(GLUT1)-mediated+glucose+metabolism+drives+a+proinflammatory+phenotype.">Metabolic reprogramming of macrophages: glucose transporter 1 (GLUT1)-mediated glucose metabolism drives a proinflammatory phenotype.</a> J Biol Chem. 289, 7884-7896 (2014).</li><li>Gonnella, R., 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=Kaposi+sarcoma+associated+herpesvirus+(KSHV)+induces+AKT+hyperphosphorylation,+bortezomib-resistance+and+GLUT-1+plasma+membrane+exposure+in+THP-1+monocytic+cell+line.">Kaposi sarcoma associated herpesvirus (KSHV) induces AKT hyperphosphorylation, bortezomib-resistance and GLUT-1 plasma membrane exposure in THP-1 monocytic cell line.</a> J Exp Clin Cancer Res. 32, 79 (2013).</li><li>Palmer, C. S., 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=Glucose+transporter+1-expressing+proinflammatory+monocytes+are+elevated+in+combination+antiretroviral+therapy-treated+and+untreated+HIV++subjects.">Glucose transporter 1-expressing proinflammatory monocytes are elevated in combination antiretroviral therapy-treated and untreated HIV+ subjects.</a> J Immunol. 193, 5595-5603 (2014).</li><li>Wong, K. L., 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=Gene+expression+profiling+reveals+the+defining+features+of+the+classical,+intermediate,+and+nonclassical+human+monocyte+subsets.">Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets.</a> Blood. 118, e16-e31 (2011).</li><li>Ziegler-Heitbrock, L., 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=Nomenclature+of+monocytes+and+dendritic+cells+in+blood.">Nomenclature of monocytes and dendritic cells in blood.</a> Blood. 116, e74-e80 (2010).</li><li>Belge, K. U., 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+proinflammatory+CD14+CD16+DR+++monocytes+are+a+major+source+of+TNF.">The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF.</a> J Immunol. 168, 3536-3542 (2002).</li><li>Frankenberger, M., Sternsdorf, T., Pechumer, H., Pforte, A., Ziegler-Heitbrock, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+cytokine+expression+in+human+blood+monocyte+subpopulations:+a+polymerase+chain+reaction+analysis.">Differential cytokine expression in human blood monocyte subpopulations: a polymerase chain reaction analysis.</a> Blood. 87, 373-377 (1996).</li><li>Ziegler-Heitbrock, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD14++CD16++blood+monocytes:+their+role+in+infection+and+inflammation.">The CD14+ CD16+ blood monocytes: their role in infection and inflammation.</a> J Leukoc Biol. 81, 584-592 (2007).</li><li>Ziegler-Heitbrock, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Macrophages: Biology and Role in the Pathology of Diseases. , 3-36 (2014).</li><li>Dimitriadis, G., 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=Evaluation+of+glucose+transport+and+its+regulation+by+insulin+in+human+monocytes+using+flow+cytometry.">Evaluation of glucose transport and its regulation by insulin in human monocytes using flow cytometry.</a> Cytometry A. 64, 27-33 (2005).</li><li>Fu, Y., Maianu, L., Melbert, B. R., Garvey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitative+glucose+transporter+gene+expression+in+human+lymphocytes,+monocytes,+and+macrophages:+a+role+for+GLUT+isoforms+1,+3,+and+5+in+the+immune+response+and+foam+cell+formation.">Facilitative glucose transporter gene expression in human lymphocytes, monocytes, and macrophages: a role for GLUT isoforms 1, 3, and 5 in the immune response and foam cell formation.</a> Blood Cells Mol Dis. 32, 182-190 (2004).</li><li>Stibenz, D., Buhrer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+L-selectin+surface+expression+by+various+leukocyte+isolation+procedures.">Down-regulation of L-selectin surface expression by various leukocyte isolation procedures.</a> Scand J Immunol. 39, 59-63 (1994).</li><li>Ahmed, N., Kansara, M., Berridge, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+regulation+of+glucose+transport+in+a+monocyte-macrophage+cell+line:+Glut-3+affinity+for+glucose+is+enhanced+during+the+respiratory+burst.">Acute regulation of glucose transport in a monocyte-macrophage cell line: Glut-3 affinity for glucose is enhanced during the respiratory burst.</a> Biochem J. 327 (Pt 2), 369-375 (1997).</li><li>Cutfield, W. S., Luk, W., Skinner, S. J., Robinson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin-mediated+glucose+uptake+in+monocytes+of+short+children+with+intrauterine+growth+retardation).">Impaired insulin-mediated glucose uptake in monocytes of short children with intrauterine growth retardation).</a> Pediatr Diabetes. 1, 186-192 (2000).</li><li>Yoshioka, 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+novel+fluorescent+derivative+of+glucose+applicable+to+the+assessment+of+glucose+uptake+activity+of+Escherichia+coli.">A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli.</a> Biochim Biophys Acta. 1289, 5-9 (1996).</li><li>Speizer, L., Haugland, R., Kutchai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+transport+of+a+fluorescent+glucose+analogue+by+human+erythrocytes.">Asymmetric transport of a fluorescent glucose analogue by human erythrocytes.</a> Biochim Biophys Acta. 815, 75-84 (1985).</li><li>Palmer, C. S., 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=Increased+glucose+metabolic+activity+is+associated+with+CD4++T-cell+activation+and+depletion+during+chronic+HIV+infection.">Increased glucose metabolic activity is associated with CD4+ T-cell activation and depletion during chronic HIV infection.</a> AIDS. 28, 297-309 (2014).</li><li>Palmer, C. S., Ostrowski, M., Balderson, B., Christian, N., Crowe, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+metabolism+regulates+T+cell+activation,+differentiation,+and+functions.">Glucose metabolism regulates T cell activation, differentiation, and functions.</a> Frontiers in immunology. 6, (2015).</li><li>Palmer, C. S., 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=Regulators+of+glucose+metabolism+in+CD4+and+CD8+T+cells.">Regulators of glucose metabolism in CD4 and CD8 T cells.</a> International reviews of immunology. , 1-12 (2015).</li><li>Palmer, C. S., Crowe, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+monocyte+metabolism+impact+inflammation+and+aging+during+chronic+HIV+infection?.">How does monocyte metabolism impact inflammation and aging during chronic HIV infection?.</a> AIDS research and human retroviruses. 30, 335-336 (2014).</li><li>McFadden, 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=Metabolic+stress+is+a+barrier+to+Epstein-Barr+virus-mediated+B-cell+immortalization.">Metabolic stress is a barrier to Epstein-Barr virus-mediated B-cell immortalization.</a> Proceedings of the National Academy of Sciences of the United States of America. 113, E782-E790 (2016).</li><li>Gamelli, R. L., Liu, H., He, L. K., Hofmann, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmentations+of+glucose+uptake+and+glucose+transporter-1+in+macrophages+following+thermal+injury+and+sepsis+in+mice.">Augmentations of glucose uptake and glucose transporter-1 in macrophages following thermal injury and sepsis in mice.</a> Journal of leukocyte biology. 59, 639-647 (1996).</li><li>Yin, Y., 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=Glucose+Oxidation+Is+Critical+for+CD4++T+Cell+Activation+in+a+Mouse+Model+of+Systemic+Lupus+Erythematosus.">Glucose Oxidation Is Critical for CD4+ T Cell Activation in a Mouse Model of Systemic Lupus Erythematosus.</a> Journal of immunology. , 80-90 (2016).</li><li>Yang, Z., Matteson, E. L., Goronzy, J. J., Weyand, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+metabolism+in+autoimmune+disease.">T-cell metabolism in autoimmune disease.</a> Arthritis research &amp; therapy. 17, 29 (2015).</li><li>Yin, Y., 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=Normalization+of+CD4++T+cell+metabolism+reverses+lupus.">Normalization of CD4+ T cell metabolism reverses lupus.</a> Science translational medicine. 7, 274ra218 (2015).</li><li>Barbera Betancourt, A., 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=Inhibition+of+Phosphoinositide+3-Kinase+p110delta+Does+Not+Affect+T+Cell+Driven+Development+of+Type+1+Diabetes+Despite+Significant+Effects+on+Cytokine+Production.">Inhibition of Phosphoinositide 3-Kinase p110delta Does Not Affect T Cell Driven Development of Type 1 Diabetes Despite Significant Effects on Cytokine Production.</a> PloS one. 11, e0146516 (2016).</li><li>Barron, C. C., Bilan, P. J., Tsakiridis, T., Tsiani, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitative+glucose+transporters:+Implications+for+cancer+detection,+prognosis+and+treatment.">Facilitative glucose transporters: Implications for cancer detection, prognosis and treatment.</a> Metabolism: clinical and experimental. 65, 124-139 (2016).</li><li>Hegedus, A., Kavanagh Williamson, M., Huthoff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+pathogenicity+and+virion+production+are+dependent+on+the+metabolic+phenotype+of+activated+CD4++T+cells.">HIV-1 pathogenicity and virion production are dependent on the metabolic phenotype of activated CD4+ T cells.</a> Retrovirology. 11, 98 (2014).</li><li>Taylor, H. 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=Phospholipase+D1+Couples+CD4++T+Cell+Activation+to+c-Myc-Dependent+Deoxyribonucleotide+Pool+Expansion+and+HIV-1+Replication.">Phospholipase D1 Couples CD4+ T Cell Activation to c-Myc-Dependent Deoxyribonucleotide Pool Expansion and HIV-1 Replication.</a> PLoS Pathog. 11, e1004864 (2015).</li><li>Loisel-Meyer, S., 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=Glut1-mediated+glucose+transport+regulates+HIV+infection.">Glut1-mediated glucose transport regulates HIV infection.</a> Proc Natl Acad Sci U S A. 109, 2549-2554 (2012).</li><li>Palmer, C. S., 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=Emerging+Role+and+Characterization+of+Immunometabolism:+Relevance+to+HIV+Pathogenesis,+Serious+Non-AIDS+Events,+and+a+Cure.">Emerging Role and Characterization of Immunometabolism: Relevance to HIV Pathogenesis, Serious Non-AIDS Events, and a Cure.</a> J Immunol. 196 (11), 4437-4444 (2016).</li></ol>

The protocol described here details a simple method to examine glucose transporter expression and fluorescent glucose analog uptake by monocyte and monocyte subpopulations in whole blood. By assessing 2-NBDG uptake in whole blood, this technique allows for conditions similar to those in vivo. A previous study examined 6-NBDG uptake in monocytes separated from whole blood by density centrifugation17. However, this study did not examine monocyte subpopulations and separation of monocytes from whole bloo...

Monocytes are a major component of the human innate immune system that are rapidly mobilized to sites of infection and inflammation1. Activation of monocytes is critical for limiting acute damage by pathogens and is also central to the pathogenesis of several chronic diseases, including atherosclerosis2, cancer3, and HIV4,5.
The metabolism of resting and activated monocytes differs dramatically, with resting monocytes utilizing oxidative metabolism and activated monocytes utilizing glycolytic metabolism (i.e., fermentation of glucose to lactate)6. Activation of monocytes induces expression of glucose transporters that allows for increased glucose uptake for glycolytic metabolism7. Monocyte glucose transporter 1 (Glut1) is one such transporter upregulated during activation and its expression has been shown to lead to production of pro-inflammatory cytokines in vitro and in adipose tissue of obese mice8. Infection of a monocytic cell line by Kaposi sarcoma associated herpesvirus leads to cellular upregulation of Glut19, and we recently showed that during chronic HIV infection an increased percentage of Glut1-expressing monocytes are present during untreated and combination antiretroviral therapy-treated infection10. Taken together, these studies show that glucose uptake and glycolytic metabolism by monocytes are important aspects of many inflammatory diseases. Thus, a simple method to measure monocyte Glut1 expression and glucose uptake during homeostasis and inflammatory disease is likely to be of use to a wide range of researchers.
Human monocytes are heterogeneous, being comprised of three distinct subsets that can be examined by differential expression of the cell surface markers CD14 and CD1611,12. Classical monocytes express a high level of CD14 but do not express CD16 (CD14++CD16-), intermediate monocytes express a high level of CD14 and an intermediate level of CD16 (CD14++CD16+), and non-classical monocytes express a low level of CD14 and a high level of CD16 (CD14+CD16++). Monocytes that express CD16 are termed CD16+ monocytes, which compared to CD16- monocytes have high expression of inflammatory cytokines and the ability to more effectively present antigens13,14. Approximately 10% of monocytes express CD16 during homeostasis with higher percentages observed during inflammation15. Monocyte subpopulations are associated with certain disease states and could be useful biological markers of disease and disease progression16.
Our goal was to identify a method that can measure glucose transporter expression and glucose uptake by human monocytes and monocyte subpopulations in conditions as close to physiological conditions as possible. Previous studies measured monocyte glucose transporter expression and glucose uptake17,18, though these methods examined isolated monocytes that can have altered protein expression compared to physiological conditions19, and no previous study has examined human monocyte subpopulations. Using multi-parametric flow cytometry, we describe a method to examine glucose transporter expression and uptake of the fluorescent glucose analog 2-NBDG by total monocytes and monocyte subpopulations (based on CD14 and CD16 expression) within whole unmanipulated blood.

<table border="0" cellpadding="0" cellspacing="0" width="570">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:140px;">VACUETT Tube 9 ml ACD-B anticoagulant tubes</td>
			<td style="width:147px;">Greiner Bio-One GmbH</td>
			<td style="width:139px;">455094</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">5 ml sterile polypropylene tubes</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">352063</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:139px;">A7906</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">16% formaldehyde solution</td>
			<td style="width:147px;">Electron Microscopy Science</td>
			<td style="width:139px;">15710</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:140px;">BD FACS lysing solution (10X)</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">349202</td>
			<td style="width:147px;">Dilute BD FACS lysing solution 1/10 with deionized water for working concentration (store for up to 1 week at 4&#176;C)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti-CD3-PE</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">555340</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti CD14-APC</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">555399</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti-CD16-PECy7</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:139px;">557744</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">anti-Glut1-FITC</td>
			<td style="width:147px;">R &#38; D Systems</td>
			<td style="width:139px;">FAB1418F</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">IgG2b-FITC</td>
			<td style="width:147px;">R &#38; D Systems</td>
			<td style="width:139px;">IC0041F</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="241">
			<td height="241" style="height:241px;width:140px;">2-NBDG</td>
			<td style="width:147px;">Life technologies</td>
			<td style="width:139px;">N13195</td>
			<td style="width:147px;">Suspend 5 mg of 2-NBDG into 1 ml of deionized water to make a 14.60 mM stock solution (keep for up to 6 months at 4&#176;C). To make the working 2-NBDG concentration, dilute stock 1/100 with 1X DPBS. Cover with foil. (store for up to 1 week at 4&#176;C)</td>
		</tr>
		<tr height="77">
			<td height="77" style="height:78px;width:140px;">Dulbecco&#8217;s Phosphate Buffered Saline (1X)</td>
			<td style="width:147px;">Life technologies</td>
			<td style="width:139px;">14190-144</td>
			<td style="width:147px;">To make wash solution, add 0.5 g BSA per 100 ml DPBS (store for up to 2 weeks at 4&#176;C)</td>
		</tr>
	</tbody>
</table>

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.

Compensation must be performed for individual fluorochromes to prevent fluorescence spillover. Monocytes are first enriched by gating based on forward and side scatter. The plots presented are representatives of at least six independent experiments conducted on whole blood from six or more participants as previously reported10. Figure 1A shows the initial gating of monocytes by cell scatter and exclusion of T cells by gating within the CD3- populatio...

Watch this Scientific Journal Video about A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood at JoVE.com

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

Research

Education

JoVE Journal

JoVE Core

Biology

Immunology and Infection

Cellular Processes

Metabolism

SCIENTISTS IN ACTION

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

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 ...

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 ...

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 ...

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

Use of a Monocyte Monolayer Assay to Evaluate FcÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Receptor-mediated Phagocytosis

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

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

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 ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...