An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Low transfection efficiency is a major obstacle to determining how protein reactivates neurite outgrowth in primary neurons. Our protocol overcome this issue by co-transfection of EGFP to identify successfully transfected cells. The high co-transfection rate of EGFP and the proteins of interest ensures the accuracy of the assay.

And consequently, immunofluorescence staining is now required. As neurite outgrowth occurs during neural regeneration, this method can be applied to the identification of novel targets for restoring the damaged neuronal network in brain trauma or neurodegenerative diseases. Before beginning the procedure, place a sterile 18 millimeter circular coverslip into each well of a 12 well tissue culture plate and coat each coverslip with five micrograms per millimeter of Poly-D-Lysine solution in a humidified 37 degree Celsius incubator for at least one hour.

At the end of the incubation, aspirate the Poly-D-Lysine solution and rinse the coverslips with sterile water. On embryonic day 18, place the uterus harvested from a timed pregnant Sprague Dawley rat into a 10 centimeter Petri dish and use small dissecting scissors to open the uterus and the amniotic sac. Use the scissors to remove the placenta and use forceps to transfer one whole embryo into a new 10 centimeter Petri dish containing chilled PBS glucose.

Use the scissors to cut along the sagittal suture of the skull and use a small flat spatula to transfer the embryonic brain into a new 10 centimeter Petri dish containing fresh ice cold PBS glucose. Using two number five tweezers and a dissection microscope, separate the two cerebral hemispheres from the cerebellum and brain stem and remove the meninges. Then isolate the cortex from the cerebral hemispheres and place the cortex in a 15 milliliter tube containing fresh ice cold PBS glucose.

For primary cortical neuron culture, in a class II biosafety cabinet, allow the cortex to settle by gravity for five minutes at four degrees Celsius before replacing the PBS glucose with 0.05%trypsin EDTA. Mix with gentle tapping and incubate the tissue in a 37 degree Celsius water bath for 10 minutes with additional gentle tapping every two minutes. At the end of the incubation, stop the digestion with four milliliters of maintenance medium and use a one milliliter pipette to dissociate the tissue with gentle trituration.

Sediment the cells by centrifugation and resuspend the pellet in one milliliter of maintenance medium for a second centrifugation. Resuspend the pellet formed after the second centrifugation in one milliliter of fresh maintenance medium for counting and plate the isolated neurons at a 6.5 times 10 to the fourth viable cells per centimeter squared concentration in one milliliter of maintenance medium per well in a 12 well plate. 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 media with the samples facing the glass slides. To image the neurite growth, select the 40X objective on an epifluorescent microscope and click start to capture images from at least 40 intact EGFP positive neurons per transfection. When all of the neurites 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. Cytochalasin D suppresses neurite extension in a dose-dependent manner while neurite outgrowth is enhanced in neurons treated with nerve growth factor. In addition, the neuronal growth adaptor protein FE65 significantly stimulates neurite outgrowth two-fold compared to untransfected neurons.

Despite a low transfection efficiency, immunofluorescence analysis reveals that over 80%of a two-day cell culture can be successfully co-transfected with EGFP and FE65. EGFP expression is detected at six hours post transfection while FE65 is first detected at hour 12 and both signals are detected for up to seven days post transfection. The transfection of primary rat cortical neurons with different amounts of FE65 plasmid DNA reveals a dose-dependent increase in neurite extension with 0 to 0.5 micrograms of FE65 plasmid.

However, no significant difference in growth is observed in neurons transfected with 0.5 or one micrograms of the plasmid. When attempting the protocol, it is important to isolate the appropriate region from the embryonic brain. This technique can also be applied to the study of neural growth in human iPSC-derived neurons which have valuable tools for regenerative medicine.