The overall goal of this experiment is to observe the real-time protein trafficking in primary neuronal cells such as GLUT4 in hypothalamic neurons with minimal damage. This method can explain key questions related to neuronal regulation of insulin signaling and metabolism such as in the case of type 2 diabetes mellitus. The main advantage of this technique is that researchers can isolate primary cell cultures and to observe the drug-induced effect in real time.
The implication of this technique is to choose a therapy of type 2 diabetes or brain-related insulin resistance because now we know how this insulin signal is being altered by the chemokine CCL5 in hypothalamic neurons. To begin this procedure coat the culture dishes with poly-D lysine one day before culture. For a six-well dish, add 1.5 mL of poly-D lysine at 0.05 mg/mL to each well.
The next day, remove poly-D lysine and wash the dishes twice with 2 mL of ddH2O. Then fill the 10-cm Petri dishes with 20 mL of wash medium and keep them on ice. Fill the 15-mL tubes with wash medium and keep them on ice.
For brain tissue isolation, sterilize the platform, dissecting microscope, and surgery tools with 75%ethanol to avoid contamination. After that, cut around the umbilical cord area to isolate the pups without damaging them. Next, remove the head of each pup and secure it in place using the fine-tipped straight forceps.
Use another fine-tipped curved forceps to remove the outer skin and skull from both sides and peel them off from the anterior to the dorsal direction. Then, keep the isolated brains in a clean Petri dish with ice cold wash medium. Flip the brain and keep the ventral side up.
Subsequently, isolate the hypothalamic tissue with fine-tipped curved forceps. Hold the olfactory lobe with the sharp forceps and peel off the thin meninges surrounding the whole brain with the curved forceps. Flip to the underside of the cortex and separate the hippocampus from the cortex by pulling it to the side.
When all regions of the brain have been isolated, chop these tissues in the 15-ml tubes containing ice cold wash medium. In this procedure, rinse the tissues by inverting the tissue-containing tube two to three times. Then, keep the tube straight to allow the tissues to settle down.
Remove the upper medium using the glass Pasteur pipette attached to a vacuum system. To wash the tissue, add 15 mL of ice cold wash medium and invert the tube two to three times. After the final wash, remove the wash medium with the help of a 1-mL pipette.
Incubate the tissues with Papain-Trypsin digestion buffer in a 37-degree Celsius water bath for seven to 14 minutes. Following that, neutralize the enzymes activity by adding one volume of fetal bovine serum. Keep the tube on rack and wait for one to two minutes to allow the tissues to settle down.
Afterward, remove the supernatant carefully with a 1-mL pipette. Add 6 mL of plating medium into the test tube and pipette the tissue up and down 50 times gently with a 5-mL pipette. Keep the tube on the rack and wait for one to two minutes to allow the chunky, undissociated tissues to settle down.
Next, transfer the upper phase containing dissociated cells to a new tube and dilute it with plating medium. Then, add 2 mL of complete culture medium into each well of the six-well dish. To prepare the cells for live imaging, carefully remove the cover slips 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 cover slip without moving it. It is essential to remove excess amount of medium to prevent unnecessary movement on the cover slip. Next, treat the selected cell samples with CCL5 at 10 ng/mL or insulin at 10 units/mL for one minute.
Following that, add 1.5 mcL of diluted insulin on the edge of the cover slips. Then, add immersion oil on a 60 times magnification 1.52 NA objective lens. Afterward, place the cover slips on the deconvolution microscope stage with the cover slips facing down and secured properly.
Use bright field 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 cover slip 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 2000 to 3000 counts to achieve maximum pixel intensity. To minimize fluorescence photo bleaching, reduce the percentage of excitation light transmission as much as possible while keeping the exposure time less than one 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.
It is very important to minimalize the fluorescent photo bleaching in neuron death following light image. This figure shows that the primary cultured hypothalamic neurons were labeled with neuron marker MAP2. The hypothalamic neuronal marker POMC and their co-expression of CCR5.
The co-localized image shows that CCL5 is expressed in POMC-positive hypothalamic neurons. These images show the expression of GFP-GLUT4 protein in wild type hypothalamic neurons and CCR5 double-negative hypothalamic neurons. Neurons were stimulated with insulin or CCL5.
The arrows point to the GFP-GLUT4 punctate in the neuritis before or after insulin or CCL5 stimulation, and asterisks indicate the surface GLUT4-GFP before or after CCL5 stimulation. Once mastered, this lab recording of primary cell technique can be done between one to four hours, depends on the experiment requirements and if it's done properly. This technique paved the way for the researchers in the field of neuroscience to explore the possibility to characterize the immediate drug-induced effect in primary cell culture.
After watching this video, you should have a good understanding how to isolate a primary hypothalamic neuron, hippocampal neuron, and a cortical neuron. Beside the lab, you can also know how to perform a live recording of the serial molecule upon different stimulations. Do not forget to put on your lab coat and gloves and frequently use ethanol to avoid contamination from bacteria and fungus from animals.
Also be very careful while dealing with cover slips and neurons throughout this procedure.
