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.
