This study investigates the role of a target protein in neurite outgrowth using primary cortical neurons. The methodology involves transfecting neurons with plasmids and analyzing the resulting neurite growth through fluorescence microscopy.
Developing neurons extend cytoplasmic processes from their cell bodies by neurite outgrowth to develop dendrites and axons, which are essential for neuron-to-neuron communication.
To study the effect of a target protein on neurite outgrowth, take a multiwell plate with a poly-D-lysine-coated coverslip at its base. The coverslip's surface has an adherent culture of primary cortical neurons derived from the embryonic rat brain.
Treat the neurons with a mix containing two types of plasmids — an EGFP plasmid encoding enhanced green fluorescent protein and a second plasmid encoding the target protein in a suitable transfection reagent.
The transfection reagent facilitates the entry of the plasmids into the cells.
Post-transfection, both plasmids get expressed, producing green fluorescent proteins and target proteins. The expression of EGFP imparts green fluorescence to the co-transfected cells.
Target proteins with neurite outgrowth-stimulating potential initiate cellular signaling, which promotes cytoskeleton reorganization and leads to the development of neuronal projections and their extensions.
Aspirate the spent medium, and fix the cells with paraformaldehyde. Counterstain the cells using red fluorescence-tagged anti-target protein antibodies that specifically bind expressed target proteins and impart additional red fluorescence to the cells.
Remove the coverslip from the culture plate, and observe it under a fluorescence microscope.
Co-transfected cells exhibiting a dual-colored fluorescence signal show longer neuronal processes than cells lacking red fluorescence, confirming the target protein's stimulating effect on neurite outgrowth.
After two days at 37 degrees Celsius and 5% carbon dioxide, dilute EGFP and target protein plasmid DNAs and transfection reagent in two separate 1.5-milliliter tubes, and then, mix them together. Transfect the cells with EGFP construct by adding the transfection mix to the wells.
After 24 hours, wash the cells with 37 degrees Celsius PBS, and fix them with 4% paraformaldehyde in PBS for 10 minutes in the dark at room temperature.
At the end of the incubation, wash the fixed cells three times with fresh PBS per wash, and add a minimal volume of fluorescence mounting medium onto one microscope glass slide, per sample. Then, carefully transfer the coated coverslips onto the mounting medium, with the samples facing the glass slides.
To image the neurite growth, select the 40x objective on an epi-fluorescent microscope, and click "Start" to capture images from at least 40 intact EGFP-positive neurons per transfection.
When all of the new rights have been imaged, open the captured images in ImageJ with the NeuronJ plugin, and measure the length of the longest neurite of each neuron from the cell body to the tip of the growth cone. Then, analyze the data obtained with the software, to determine the effect of the targeted proteins on neurite growth.
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