The overall goal of this procedure is to introduce genes and short hairpin RNAs into cultured neurons. This is accomplished by first establishing astroglia cultures from neonatal rats to support the neuronal growth. Hippocampal neurons from E 18 rats are then co cultured with the glial cells.
The next step is to transfect the hippocampal neurons using a calcium phosphate precipitation method, the transfected neurons are then visualized by fluorescence microscopy. Using these methods, the transfection efficiency can be up to 50%The main advantage of this technique over existing methods is it is relatively simple, economical, and very efficient. Though our protocol focuses on the transfection of cultured hippocampal neurons, it can also be applied to other difficult to transfect cell lines.
For this protocol, have prepared BSS dissection buffer at four degrees Celsius ready for use. After decapitating a pup, hold the head firmly with a pair of number five forceps and use fine scissors to make a midline incision through the skin and skull. Expose the brain by reflecting the skull to the sides.
Then remove the brain into a dish containing cold BSS. Under a dissecting scope, separate the cerebral hemispheres from the cephalon and the brainstem. Carefully remove all the meninges by stabilizing the tissue with one pair of forceps and gently pulling away the meninges with another pair of forceps.
Using small scissors, mince the tissue as finely as possible. Then transfer the minced tissue to a 50 milliliter conical tube. Add BSS to the dish with the remaining tissue, and then transfer the mixture to a conical tube.
Add DNAs one and trypsin to a total volume of 15 milliliters. Incubate the tissues in a shaking water bath for 15 minutes at 37 degrees Celsius. To ensure good mixing, swirl the tube by hand every five minutes.
Allow the tissue to settle. Then pass the supernatant through a 70 micron cell strainer into a 50 milliliter conical tube containing three milliliters of FBS to the remaining pieces. Add more B, s, s and trypsin.
Then incubate them for 15 more minutes. Transfer the remaining supernatant through the cell strainer and combine it with the other supernatant. Now centrifuge the combined collection at 1000 RPM for five minutes.
Reus, suspend the cell pellet in five milliliters of glial media. After counting the cells plate 10 million cells for each 150 square centimeter flask and begin the incubation the next day. Replace the media with fresh glial media.
Thereafter, feed the cells twice a week with glial media. The cells will reach more than 80%co fluency after about 10 days. At this time, freeze the cells in serum with the MSO at 2 million cells per vial.
Freeze the aliquots in a styrofoam rack at minus 80 degrees Celsius before moving them to liquid nitrogen for long-term storage. 10 to 14 days before setting up the hippocampal culture plate, the glial cells from the frozen stocks. Five six.
Well plates are enough to support the growth of 90 cover slips of hippocampal neurons plate an additional eight to 10 60 millimeter dishes of glial cells. To condition the N 2.1 media for transfection the day before neuronal culture. Feed the cells in B 27 media.
The astrocytes should be about 90%confluent at this point after euthanizing a pregnant rat at stage E 18 by isof fluorine. Anesthesia followed by pneumothorax. Dissect out an embryonic rat.
Then remove the embryonic brain into BSS. Isolate the cerebral hemispheres ears, and remove the meninges from the cerebral hemispheres. Next, dissect out the hippo campi from the cerebral hemispheres.
Transfer all the hippo campi to a single 15 milliliter conical tube and digest them in 0.25%trypsin in BSS at 37 degrees Celsius for 15 minutes. Next, wash the digested hippo campi in BS S3 times for five minutes per wash. Then iterate the hippo campi with a flame polished glass pesi pipette.
After counting the neurons plate out 200, 000 cells per 60 millimeter dish. Two to four hours after plating. Transfer the cover slips with the attached neurons to the astro Cleal cultures.
Place the neurons facing down towards the ggl at div three. Stop the ggl proliferation by adding Cytosine Arab side as described earlier on the day before the transfection aspirate the media from the glial dish and add N 2.1 media for conditioning the next day. Harvest the media from the glia and transfer the media to a new dish.
Then transfer the cover lips with neurons into the conditioned N 2.1 media. Let them equilibrate in an incubator for 10 to 30 minutes. In a set of sterile tubes, combine one to four micrograms of DNA 12.5 microliters of two molar calcium chloride, and sterile water.
For a total volume of 100 microliters. Load a second set of tubes each with 100 microliters of two XHBS. Then gradually add two XHBS from one tube to 1D NA mixture tube in one eighth volumes.
Vortex the mixture for a few seconds between each edition. After the last edition, allow the tubes to sit for 15 minutes. After 15 minutes, add each prepared transfection mixture.
Dropwise to a plate of cells, incubate the cells for one to one and a half hours. A layer of sand like precipitates should form it is visible using a 10 x or 20 x objective. After the incubation, rinse the cover slips twice with warm HBS wash buffer.
Then return the cover slips to the original dishes containing glia. Now add reic acid to the cover slips to a final concentration of one half millimolar. The transfected neurons can be imaged live or further processed on the next day.
However, they will survive on the cover slips for up to three more weeks. When the different parameters of transfection are optimized, it is possible to obtain efficiencies of up to 50%These neurons were transfected with GFP on div four, and a transfection efficiency of over 50%was typical for any view of the culture. Neurons in the culture remained healthy for a fair length of time.
At DIV 15, 10 days after transfection with a PSD 95 GFP construct, hippocampal neurons showed numerous mature mushroom shaped dendritic spines, a strong indication of healthy neurons While attempting this procedure, it's important to remember to keep all procedures and reagents as consistent as possible to achieve reproducibility.
