Immunology and Infection
Published: August 12th, 2016
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 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 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 induce....
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
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.......
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.......
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 Progr....
|VACUETT Tube 9 ml ACD-B anticoagulant tubes
|Greiner Bio-One GmbH
|5 ml sterile polypropylene tubes
|Albumin from Bovine Serum (BSA)
|16% formaldehyde solution
|Electron Microscopy Science
|BD FACS lysing solution (10X)
|Dilute BD FACS lysing solution 1/10 with deionized water for working concentration (store for up to 1 week at 4°C)
|R & D Systems
|R & D Systems
|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°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°C)
|Dulbecco’s Phosphate Buffered Saline (1X)
|To make wash solution, add 0.5 g BSA per 100 ml DPBS (store for up to 2 weeks at 4°C)
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