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. 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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. 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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. 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The authors have nothing to disclose.

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>
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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...

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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 ...

The overall goal of this methodology is to measure the uptake of glucose and its transporter on different monocyte subpopulations. This method can be used to answer key questions in the immunology field, such as What is the importance of immune cell metabolism in disease outcomes? The main advantages of these techniques are it is quick, easy to perform, requires no reactive material, and requires only a small volume of blood.Within one hour of collecting the blood samples, in citrate ACD-B anticoagulant tubes, place the samples into a biological safety cabinet. Next, add 100 microliters of blood from each sample into individual polypropylene tubes, and treat the cells with two milliliters of 1X lysing solution on ice, pipetting gently to mix. After 15 minutes, centrifuge the cells and follow this with two more spins in wash buffer.After the second wash, use a pipette to carefully remove as much of the supernatants as possible, and re-suspend the pellets in 100 microliters of wash buffer on ice. To identify the specific monocyte subpopulations, stain the cells with five microliters of the appropriate antibodies per 100 microliters of cells, for 30 minutes on ice in the the dark. At the end of the incubation, wash the samples two times in wash solution and fix the cells with 200 microliters of 0.5%formaldehyde in PBS.Then, analyze the cells within 24 hours on a flow cytometer capable of detecting four colors. To assess the glucose uptake of the monocyte subpopulations, transfer 90 microliters of blood from each sample into individual polypropylene tubes, and add 10 microliters of the fluorescent glucose analog of interest to the blood. Flick the tubes gently to mix, and cover the samples with aluminum foil.Then, place the cells at 37 degrees Celsius for 15 to 30 minutes, followed by their immediate transfer to ice. Next, add four milliliters of FACS lysing solution to the tubes, and centrifuge the cells. Then, perform a second centrifugation in wash buffer.Decant the supernatant, and stain the cells with the appropriate antibodies of interest for 30 minutes at four degrees Celsius in the dark. Then, wash the samples in four milliliters of ice-cold wash buffer, re-suspend the pellets in 200 to 300 microliters of ice-cold PBS, and analyze the tubes within 10 minutes. For flow cytometric analysis, first use the unstained and individually stained control samples to set the compensation.Next, in the appropriate single-cell analysis software, open the first sample in a forward by side scatter plot, and gate the monocytes as illustrated. Then, double-click the gated monocyte population, and gate the CD3-negative population. Now double-click on the CD3-negative population, and set the x-axis to display the CD14 staining, and the y-axis to display the CD16 staining.Next, to measure the uptake of glucose or its transporter in a specific monocyte subpopulation, generate the appropriate histograms. Then, where defined cell populations exist, use the control FITC igG samples to set the gate and determine the total percentage of CD14-positive monocytes in each sample. Here, the initial gating strategy for isolating the monocytes by cell scatter and T-cell exclusion by gating within the CD3-negative population, is shown.The monocytes are then gated for CD14 expression alone, or in combination with CD16 to identify the total monocytes or monocyte subpopulations. Distinct populations of CD14-positive Glut-1 glucose transporter positive monocytes may be observed, most notably in the circulating white blood cells obtained from HIV-positive individuals. CD14-positive Glut-1 positive cells may also be observed within specific monocyte subsets in HIV-uninfected individuals, although these populations are more pronounced in HIV-positive patients.As expected from the glucose transporter expression data, the glucose analog 2-NBDG, is also taken up in higher quantities by cells from HIV-positive individuals. Once mastered, this technique can be completed within one-and-a-half hours if performed properly. While attempting this procedure, it is important to limit light exposure to all regions.Following glucose analog uptakes, cells can be stained with cell surface antibodies to identify specific immune populations that you're interested in. After its development, this method can be used to determine how HIV affects immune cell metabolism, can also be explored by other researchers, in different fields, such as immunology, virology, and cancer research. After watching this video, you should have a good understanding on how to detect glucose transporter one, and glucose uptake in immune cells.Don't forget that when working with infectious agents, standard safety procedures should be employed.

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16.3K Views.  Macfarlane Burnet Institute for Medical Research and Public Health. The overall goal of this methodology is to measure the uptake of glucose and its transporter on different monocyte subpopulations. This method can be used to answer key questions in the immunology field, such as What is the importance of immune cell metabolism in disease outcomes? The main advantages of these techniques are it is quick, easy to perform, requires no reactive material, and requires only a small volume of blood.Within one hour of collecting the blood samples, in citrate A...