Name: Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry (Video) | JoVE Journal
Uploaded: 2010-11-07T17:00:00.000+00:00
Duration: 5 min 39 s
Description: Learn to quantify GLUT4 translocation to the plasma membrane using flow cytometry in insulin-stimulated myoblasts. This method enables rapid detection of glucose transporter dynamics for metabolic disease research.

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5 minutes

This work was supported by grants from the Mrs. Clifford Elder White Graham Endowed Research Fund &amp; the NIH (AR059838) to CB, from the NIH (AR052791, AR044387-ARRA, &amp; NS063298) to LTT, and by the Baylor College of Medicine Cytometry and Cell Sorting Core with funding from the NIH (NCRR S10RR024574, NIAID AI036211, &amp; NCI P30CA125123). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

1. 	Cell Staining
<ol>
<li>Prepare the cells. The cells should be used while still actively growing (60- 80% confluence). Serum starve the cells overnight, plate them cells at 0.1 million per well in 0.5 mL serum-free medium in a 24-well plate and place in the incubator (37°C, 5% CO2) for 30 min - 2 hours to recover from trypsinization.</li>
<li>Mix the primary and secondary antibodies. Mix (5 μL of primary anti-GLUT4 antibody with 1 μL secondary chicken anti-goat IgG antibody conjugated to AlexaFluor 488) x number of wells. Incubate for 10 minutes at room temperature, in the dark.</li>
<li>Prepare a 2X working stock of insulin by diluting it in medium.</li>
<li>Check if the cells have adhered or not.</li>
<li>Gently add 6 μL antibody mix and 0.5 mL of your 2X working stock of insulin to the wells containing the cells. Avoid any turbulence of the medium. Incubate (37°C, 5% CO2) for 30 minutes, in the dark.</li>
<li>Fix the cells by adding 0.5 mL of PBS + 1% PFA to each well, without shaking the cells. Incubate for 20 minutes at room temperature, in the dark.</li>
<li>If your cells have adhered to the wells, gently lift them with a cell lifter. Transfer the cells (1 mL total) into tubes that fit in your flow cytometer and centrifuge to pellet the cells.</li>
<li>Wash the pellet twice with 1 mL PBS and resuspend in 0.4 mL PBS + 1% PFA. At this point, cells can be wrapped in foil and stored in the fridge (4-8°C) or taken to the flow cytometer for immediate data acquisition.</li>
</ol>
2. 	Data Acquisition
<ol>
<li>Set a broad gate around the live cells in the side scatter (SSC) versus forward scatter (FCS) panel. Use a negative tube (cells not treated with insulin or cells "stained" with an isotype control) to set your FL1 parameter. Make sure your peak is completely in the panel, not cut on the lower side.</li>
<li>Acquire data from at least 10,000 cells per tube.</li>
</ol>
3. 	Data Analysis
<ol>
<li>Draw a gate around the live cells in the SSC vs FSC panel (Figure 1A). Plot the histograms of AlexaFluor 488 fluorescence intensity versus number of cells within the live-cell gate (Figure 1B). You can overlay different histograms to visualize differences but for quantitation, determine the mean and median fluorescence intensity for each cell population and use these numbers for your comparisons (Figure 1C).</li> 
<li>Normalize the data to the value obtained in the absence of insulin, as shown in Figures 1C and 1D.</li>
</ol>
4. Representative Results
In myoblasts isolated from a healthy individual (no insulin resistance), insulin induces a dose-dependent translocation of GLUT4 from the cytoplasm to the plasma membrane (Figure 1, left). In contrast, insulin does not induce GLUT4 translocation to the plasma membrane of myoblasts isolated from a patient with insulin resistance (Figure 1, right).
<img src="/files/ftp_upload/2429/2429fig1.jpg" alt="Flow cytometry analysis of insulin resistance; scatter plots, histograms, and MFI bar charts analyze cells." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Example of a staining experiment. Myoblasts were isolated from a donor without insulin resistance (left) and from a patient with insulin resistance (right). A, SSC (side scatter) versus FCS (forward scatter) plot with a gate around the live cells. B, Cells gated in A, stained with the antibody against an external epitope of GLUT4, and left unstimulated (red) or stimulated with insulin 1 nM (blue), 10 nM (orange), or 100 nM (green). For figure clarity, we only show data without and with 100 nM insulin for the cells with insulin resistance. C, Mean fluorescence intensities (MFI) of the histograms shown in B were normalized to the MFI of the cells left unstimulated (no insulin). D, Plots of the normalized MFI data from the histograms shown in B.

<ol><li>Zhao, F. Q., Keating, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+properties+and+genomics+of+glucose+transporters%5BTitle%5D)+AND+%22Curr.+Genomics%22%5BJournal%5D)">Functional properties and genomics of glucose transporters.</a> Curr. Genomics. 8, 113-128 (2007).</li>
<li>Karnieli, E., Armoni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+regulation+of+the+insulin-responsive+glucose+transporter+GLUT4+gene%3A+from+physiology+to+pathology%5BTitle%5D)+AND+%22Am.+J.+Endocrinol.+Metab%22%5BJournal%5D)">Transcriptional regulation of the insulin-responsive glucose transporter GLUT4 gene: from physiology to pathology.</a> Am. J. Endocrinol. Metab. 295, 38-45 (2008).</li>
<li>Graham, T. E., Kahn, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-specific+alterations+of+glucose+transport+and+molecular+mechanisms+of+intertissue+communication+in+obesity+and+type+2+diabetes%5BTitle%5D)+AND+%22Horm.+Metab.+Res%22%5BJournal%5D)">Tissue-specific alterations of glucose transport and molecular mechanisms of intertissue communication in obesity and type 2 diabetes.</a> Horm. Metab. Res. 39, 717-721 (2007).</li>
<li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">CD107a as a functional marker for the identification of natural killer cell activity.</a> J. Immunol. Methods. 294, 15-22 (2004).</li>
<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutellingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J. Immunol. Methods. 184, 39-51 (1995).</li>
<li>Lugli, E., Roederer, M., Cossarizza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Data+analysis+in+flow+cytometry%3A+the+future+just+started%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Data analysis in flow cytometry: the future just started.</a> Cytometry A. 77, 705-713 (2010).</li>
</ol>

No conflicts of interest declared.

We have demonstrated a rapid technique for quantifying the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.
GLUT4 is only transiently expressed at the plasma membrane of cells and is endocytosed. Since the bound antibodies remain attached to the GLUT4, even during endocytosis, the fluorescence signal gives the total amount of GLUT4 that was exposed at the plasma membrane during the chosen duration of incubation in the presence of insulin.
This technique can also be applied to studying the translocation of other proteins from an intracellular compartment to the plasma membrane, provided a good antibody directed to an external epitope of the protein of interest is available. For example, a similar assay is now commonly used to assess the rate of degranulation of cytotoxic T lymphocytes and natural killer lymphocytes by measuring the translocation of CD107a to their plasma membrane4,5. The translocation of phosphatidylserine from the inner side of the plasma membrane to the outer side during cell apoptosis or necrosis can also be detected by flow cytometry, using fluorophore-labeled Annexin V6.
Besides rapidity of the assay, flow cytometry presents the advantage of allowing the study of protein translocation to the plasma membrane in mixed cell populations. Indeed, one can combine staining for cell subset markers and for the protein of interest, followed by data acquisition on a multi-laser flow cytometer7.
The amounts of antibodies given in paragraph 1.2 of the protocol are a starting point; we recommend you titrate your antibodies with your cells of interest to determine the minimum antibody concentration that gives you a maximum signal. A good starting range for the primary anti-human GLUT4 antibody we have used here would be 1-10 &mu;L per tube (i.e. 0.2-2 &mu;g antibody per tube), each tube containing no more than 1 million cells. For other primary antibodies, refer to the supplier's instructions.
Normalization of data is necessary as autofluorescence of cells will vary from donor to donor and from experiment to experiment.

<table><tbody><tr><td>Anti-human GLUT4 antibodies</td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-1606</td><td>Against an external epitope of GLUT4</td></tr><tr><td>Chicken anti-goat IgG conjugated to AlexaFluor 488</td><td>Invitrogen</td><td>A-21467</td><td></td></tr><tr><td>Recombinant human insulin</td><td>Sigma-Aldrich</td><td>I2643</td><td></td></tr><tr><td>Cell lifters</td><td>VWR international</td><td>29942-118</td><td>Cut to fit the wells</td></tr><tr><td>5 ml tubes for FACS</td><td>VWR international</td><td>60819-138</td><td>Fit all BD machines</td></tr></tbody></table>

quantitative measurement of glut4 translocation to the plasma membrane by flow cytometry

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

The overall goal of this procedure is to detect glute four translocation to the plasma membrane of mammalian cells in response to in insulin exposure. Serum starved myoblasts, which possess only intravesicular GLUT four protein are first plated in a 24 well cultured dish antibody against an external epitope of glut. Four pre incubated with a Fluor conjugated secondary antibody is then added to the cells, followed by insulin.Engagement of the insulin receptors at the surface of the cells results in translocation of glute four to the plasma membrane surface. Glute four is then recognized by antibodies in the medium. At this point, the cells can be fixed and the intensity of fluorescent staining measured by flow cytometry.This method can be used to study ggl four translocation and glucose metabolism, but also can be applied to other systems such as NK cell degranulation through exposure of CD 1 0 7 A at the membrane. For this procedure, it is important to use cells that are actively growing or 60 to 80%cofluent and have been serum starved overnight. After harvesting the cells by trypsin, wash and resuspend them in regular growth medium at 200, 000 cells per milliliter plate, the cells in a 24 well plate at 0.1 million per 500 microliters per well Then incubate the cells for one hour at 37 degrees Celsius to allow cells to recover from trypsin.In the meantime, mix the primary anti glute four antibody and the Alexa Fluor 4 8 8 conjugated secondary chicken antigo IgG antibody in a five to one ratio, calculate the final volume needed, taking into account that 10 microliters of the antibody mix will be used for each. Well incubate the antibody mix for 10 minutes of room temperature in the dark while the antibody mixture is incubating. Prepare a two times working stock of insulin by diluting it to the desired concentration in growth medium.Once the cells have incubated for one hour, remove them from the incubator. Gently add 10 microliters of antibody solution to each well containing cells. Be sure to leave at least one well unstained.This negative sample will be used later to set up the flow cytometer. Next, add 500 microliters of the two times insulin stock to each well to trigger translocation of glute four to the cell surface and incubate the plate for 30 minutes at 37 degrees Celsius in the dark. After removing cells from the incubator, fix them by adding one milliliter of PBS 1%PFA to each well and incubate the plate for 20 minutes At room temperature in the dark, using a cell lifter gently detach the fixed cells from the bottom of the wells and transfer them from each well to an individual fax tube.Centrifuge the tubes to pellet the cells, then vortex and resuspend them in one milliliter of PBS to wash and centrifuge the cells again, repeat the wash. Step twice. Discarding the supernatant between washes.Finally resuspend the wash cells in 400 microliters of PBS 1%PFA. The cells can now be analyzed for surface expression of glute four in the flow cytometer. Once you've arrived at the flow cytometer with your samples, begin acquiring data from the unstained control.Define a broad live cell gate using the forward and side scattered parameters. Then adjust the voltage setting for the FL one channel so that the negative peak is visible within the margins of the histogram. Once these parameters have been set, run each experimental sample recording at least 10, 000 live events per tube.This histogram shows the upregulation of surface glut force staining or myoblasts exposed to 0 1 10 or 100 nanomolar insulin. In contrast, myoblasts from insulin resistant individuals fail to upregulate glu four in response to 100 nanomolar insulin. If the mean fluorescent intensities of stimulated cells is normalized to that of unstimulated cells, it is possible to compare the healthy and insulin resistant myoblast MFIs directly.Using this procedure, cells can be stained for additional surface or intracellular markers to identify different cell subsets in mixed populations.

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Learn to quantify GLUT4 translocation to the plasma membrane using flow cytometry in insulin-stimulated myoblasts. This method enables rapid detection of glucose transporter dynamics for metabolic disease research.

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

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