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.
