The overall goal of this procedure is to isolate and culture the cortical or hippocampal neurons in serum free conditions. This is accomplished by first dissecting the rat embryos to obtain the cortices or hippo campi. The second step is to incubate the brain tissue with trip le express for gentle enzymatic digestion.
Next, the tissues will be rinsed with hibernate E and mechanically triturated and complete culture medium. To obtain single cell suspension, the final step is to count and plate the dissociated neurons on poly de lysine coated dishes or poly de lysine, laminin coated glass slides. Ultimately, this method allows the isolation and culture of the neurons in serum free conditions and without glial support.
Neuronal cultures can be used for immunochemistry nucleic acids, preparation and electrophysiology. The main advantage of this technique over existing methods is that we have replaced trypsin with a gentler enzyme. We have omitted the means of the tissue before enzymatic digestion, as well as the use of DN a's treatment.
Also, the dissected Cortes or hippo can be stored in hibernate medium at four degrees for up to a week before their dissociation. To prepare PDL solution first, add five milliliters of sterile double distilled water to five milligrams of PDL to obtain a stock solution of one milligram per milliliter. Mix the stock solution by pipetting it several times, then use it immediately or store it at two to eight degrees Celsius.
To coat the culture dishes. Dilute the PDL stock solution with sterile double distilled water to the final concentration of 10 micrograms per milliliter. Next pipette three milliliters solution into a 60 millimeter dish to cover the culture surface area.
After that, rock the dish to ensure even coating of the culture surface. Then incubate it at room temperature overnight. On the next day, remove the PDL solution by aspiration.
Briefly, wash the dish twice with three milliliters of sterile double distilled water. After the second wash, remove the water completely by aspiration. In this procedure, mix the PDL stock solution of one milligram per milliliter and laminate stock solution of one milligram per milliliter in sterile double distilled water to the final concentration of 10 and five micrograms per milliliter respectively.
Next, pipette enough solution into the wells of a glass two chamber slide to cover the culture surface. Then rock the chamber gently to ensure even coating. After that, incubate the coated dish at room temperature overnight.
On the next day, remove the PDL laminate coating solution by aspiration. Briefly, wash the dish twice with one milliliter of sterile double distilled water After the second wash. Remove the water completely by aspiration in this step, warm trip LE express and neuro basal B 27, complete medium in a 37 degree Celsius water bath.
Then add three milliliters of cold hibernate E solution to each of the four 60 millimeter culture dishes and 13 milliliters of it to a 15 milliliter BD Falcon High clarity polypropylene conical tube. Afterward, add 25 to 30 milliliters of cold dissection medium to each of the three 100 millimeter culture dishes for washing the embryos immediately after their removal from the amniotic sax. Next, sacrifice an E 17 timed pregnant rat and spray its lower abdomen with 70%ethanol.
Then cut medially through the skin and muscles with a pair of scissors. To expose the uterus and embryos, remove all the fetuses and place them in a sterile 100 millimeter dish containing an excess of cold dissection. Medium prepared earlier.
Afterward, cut the embryos from the amniotic sack using a pair of small scissors. Then place them in the second 100 millimeter dish containing the cold dissection medium. Wash the embryos at room temperature by gently tilting the dish for five to 10 seconds.
Subsequently, transfer the rinsed embryos to the third 100 millimeter dish containing the dissecting medium Under a stereo microscope, extract each rat embryo's brain by pulling out the skin and skull with a pair of curved forceps. Place about five brains in a 60 millimeter dish with cold hibernate E.Then keep the dishes on ice. Take one dish at a time under the dissecting microscope.
Separate the hemispheres and isolate the cerebral cortices. Then remove the midbrain and meninges. Collect all the dissected cortices in a 15 milliliter clear conical tube containing 13 milliliters of cold hibernate.
E.Leave the cerebral cortices on ice until all the dissections are completed. Transfer the tube to a tissue culture hood. Then allow the cortices to settle at the bottom of the tube and carefully remove the supra natin.
Next, add 13 milliliters of fresh hibernate E to the 15 milliliter conical tube. Allow the cortices to settle at the bottom of the tube and carefully remove the supernatant. Again, repeat these procedures two more times and after the last wash, carefully remove all media.
Next, digest the cerebral cortices by adding one to two milliliters of warm trip le express. Seal the cap of the tube with paraform. Then place the tube in a 37 degree Celsius water bath for 10 minutes.
After that, spray the tube with 70%ethanol. Then add 10 milliliters of hibernate E.Allow the cortices to settle at the bottom of the tube and remove the supernatant. Repeat this step three times to wash out the trip Le express.
And in the last step, carefully remove all the media. Gently tritrate the cortices about four to five times in two milliliters of neuro basal B 27 complete medium. Using a fire polished glass pasture Pasteur tritrate the cortices for another four to five times with a sterile glass pasteur pipette with smaller diameter.
Then allow the remaining pieces of tissue to settle. After that, transfer the upper single cell suspension to a new 15 milliliter tube leaving behind the settled pieces of tissue. Then further dilute the cell suspension up to 10 to 12 milliliters with neuro basal B 27, complete medium to dilute the cells for counting.
Add 10 microliters of cell suspension to 490 microliters of 50 x counting solution in a 1.5 milliliter eend tube plate. The cells on the PDL coated plates at a density of 50, 000 cells per square centimeter. Neurons can be used for experiments after four to five days in vitro.
Shown here is a representative image of a cortical neuron nucleo affected with P max GFP and immuno labeled with red map two antibody. And here shows the five days in vitro culture of rat cortical neurons isolated from the cortices, which had been kept at four degrees Celsius for one week in hibernate E plus B 27 after their original dissection from the embryos. Neurons were plated on a glass two chamber slide coated with PDL and laminin as previously described.
The green immunofluorescence indicates the expression of map two in neuronal processes and the cellular nuclei are indicated in blue. Once master, this technique can be done in about two hours if it's performed properly. After watching this video, you should have a good understanding of how to isolate and culture neurons in serum free, Anglia free conditions.
