This study investigates the role of CCR5 in the translocation of glucose transporter-4 (GLUT4) in hypothalamic neurons. Using wild-type and CCR5-knockout mouse neurons, the effects of insulin and CCL5 on GLUT4 membrane translocation were analyzed through live imaging techniques.
Begin with wild-type and CCR5-knockout mouse primary hypothalamic neurons cultured on poly-D-lysine-coated coverslips in a multi-well plate.
These neurons express green fluorescent protein-tagged glucose transporter-4 (GFP-GLUT4) in the cytoplasm.
Transfer the coverslips onto glass slides and gently wipe off excess medium to ensure stability during image acquisition.
Place a slide on the deconvolution microscope stage with the coverslip facing downward.
Visualize the neurons under bright-field illumination, then treat them with the chemokine CCL5 and insulin sequentially.
Switch to fluorescence illumination to identify GFP-GLUT4-expressing target neurons and adjust the imaging parameters to capture live fluorescent images.
In wild-type neurons, insulin and CCL5 bind to the insulin receptor and CCR5, respectively, activating downstream signaling pathways that drive GFP-GLUT4 membrane translocation.
In CCR5-knockout neurons, CCL5 fails to bind, resulting in reduced GFP-GLUT4 translocation.
Capture the images and apply a deconvolution algorithm to reduce out-of-focus light, enhancing image clarity and resolution.
To prepare the cells for live imaging, carefully remove the coverslips with neurons using forceps and place them on the glass slide with caution. Then remove excess medium with delicate task wipes by folding them twice and carefully placing them on the coverslip without moving it.
It is essential to remove excess amount of medium to prevent unnecessary movement on the coverslip.
Next, treat the selected cell samples with CCL5 at 10 nanograms per milliliter or insulin at 10 units per milliliter for one minute. Following that, add 1.5 microliters of diluted insulin on the edge of the coverslips. Then, add immersion oil on a 60 times magnification 1.52 NA objective lens.
Afterward, place the coverslips on the deconvolution microscope stage with the coverslips facing down and secured properly. Use brightfield or fluorescence illumination to identify the target cells. Afterward, adjust the focus until the target cells can be clearly observed. Do not move the objective lens outside of the coverslip area to avoid unnecessary scratch marks.
Next, identify GFP-GLUT4 by the GFP signal. Then identify the desired target cells for image acquisition. Subsequently, set the proper experimental parameters on each target cell, including pixel number, excitation wavelength, transmission percentage, exposure time, stack thickness, time interval, and total imaging time. Adjust the exposure parameter to approximately 2,000 to 3,000 counts to achieve maximum pixel intensity.
To minimize fluorescence photobleaching, reduce the percentage of excitation light transmission as much as possible, while keeping the exposure time less than 1 second. Set the upper and lower limit of the z-stack on each target cell by moving the microscope stage until the top and bottom of the target cell are both slightly out of focus.
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