The goal of this procedure is to culture primary neurons in the presence of a layer of glia that support their growth. These neurons can later be isolated as a pure population of neurons for analysis. This is accomplished by first culturing the primary glial cells.
The second step is to subculture the glial cells onto a surface, which will oppose the cultured neurons. Then the neuronal glial cell layers are combined into a stable co-culture system. Ultimately, cyto chemical analysis shows the development of a pure layer of mature neurons.
Hi, my name is Anna T and I'm a student at Dr.Olympia Muci lab at Drexel University. Here we'll demonstrate how to culture at cortical neurons in the presence of a glia feed layer. This is a technique that was originally developed by Gary Banker and colleagues and learned by Dr.Muci and Richard Miller's laboratory at the University of Chicago, which we have then adapted to our experimental needs.
The main advantage of this technique over pure neuronal culture is that the trophy factors released by glia support neuronal growth and differentiation. While the neurons can later be isolated as a pure population. To prepare for the dissection place sterile tools in 70%ethanol, add four milliliters of cold dissection, medium to 60 millimeter dishes, then thaw 2.5%trips in and DNAs on ice.
After decapitating the two to four day old pups, place the heads in a 100 millimeter sterile dish for each head. Make a midline cut through the skin and pull the skin back in order to expose the skull. Then use a pair of curved scissors to make a midline cut and two lateral cuts through the skull without damaging the brain tissue underneath.
Following this, remove the upper skull to expose the brain, then extract the brain and place it in the 60 millimeter dish with four milliliters of cold dissection, medium prepared earlier under a stereo microscope. Separate the hemispheres, remove the midbrain and peel off the meninges. Collect all of the dissected cerebral cortices in a new 60 millimeter dish with fresh cold dissection medium.
Then mince the tissue with a pair of curved scissors. Subsequently transfer the mince tissue into a 15 milliliter sterile tube. Bring the volume to 4.5 milliliters with cold dissection medium and add 500 microliters of 2.5%trips in.
Then wrap the tube in para film and place it in a 37 degrees Celsius water bath for 15 minutes. In the meantime, mix it by inverting the tube every five minutes. Next, transfer the tissue to a new 15 milliliter tube and try to minimize the tripsy containing dissection medium being transferred to the tube.
Now bring the final volume to 4.8 milliliters with fresh dissection medium, and add 200 microliters of DNAs to a final concentration of 60 micrograms per milliliter. Then triturate gently with a sterile five milliliter pipette about 20 times, and add 10 milliliters of glio plating medium. Allow four to five minutes for the large pieces of tissue to settle at the bottom.
After that, transfer the top 80%of the cell suspension to a 50 milliliter tube. Then repeat the tissue duration with an additional two to three milliliters of GL plating.Medium. Again, allow the tissue to settle.
Transfer the top 80%to the previous collection afterwards. Bring the total volume to 20 milliliters. Next, centrifuge it for 15 minutes at 280 times G.After that, aspirate the supinate and resuspend the cell palette in five to seven milliliters of ggl plating medium.
Then add additional plating, medium to 10 milliliters for each brain dissected and mix the cells well. Plate 10 milliliters of cell suspension in each 75 centimeter square flask. Following this, incubate the culture flask at 37 degrees Celsius.
Change the culture medium after 24 to 48 hours, then every three to four days to prepare Therman cover slips. The plastic cover slips used a culture secondary ggl add three small drops of melted para plast wax around the circumference of each washed and autoclave thermadox. The cells will be plated on this side of the thermadox.
Our thermon are handmade, reusable 60 millimeter inserts with about 52 millimeter holes drilled through. Although commercially available culture well inserts may also be used. The next step is to wash the primary ggl with PBS twice.
Shake each flask vigorously to remove any unattached cells. Each flask with confluent primary ggl will provide roughly 10 million astroglia. Therefore, we often use two to four flasks per experiment.
Next, detach the cells with 0.05%trips in EDTA. Combine the cells from two flasks into one 50 milliliter conical tube and add an equal amount of ggl plating medium, then centrifuge for 15 minutes at 280 times gravity. Aspirate the supinate and dissolve the GL pellets in a few milliliters of GL plating.Medium.
Count the cells using a hemo cytometer and dilute the cell suspension as needed. Then plate 500, 000 cells either on 60 millimeter dishes or on therman cover slips using GL plating medium for thermon plate. 600 microliters of cells on each cover slip in a dropwise fashion, and after two hours, flip each thermadox to submerge the cells in glia plating medium.
If preparing 60 millimeter dishes of secondary glia for neuronal cover slips, change the medium to end two the evening before the neuronal dissection. In this procedure, to prepare the 15 millimeter glass cover slips, add three small drops of melted para blasts. Wax around the circumference of each sterile cover slip in order to physically separate the cell layers and prevent cell scraping coat.
The cover slips all 60 millimeter dishes with 0.5 milligrams per milliliter polylysine for two hours or overnight. Then wash them with sterile water two to three times and allow them to dry completely To prepare the neuronal dissection. Euthanize a 17 day pregnant rat.
Spray it with 70%ethanol and cut medially through the skin to expose the uterus. After that, remove all fetuses and place them in a sterile 100 millimeter dish. After rapidly dissecting and decapitating each fetus, place each head in a 60 millimeter dish with four milliliters of cold dissection medium under the stereo microscope.
Use a pair of forceps to isolate the cerebral cortices by pulling open the skin and skull, extracting the brain, separating the hemispheres and removing the midbrain and meninges. Collect all the dissected cortices in a fresh 60 millimeter dish with cold dissection medium. Then mince the tissue with a pair of curved scissors into around one millimeter pieces.
Transfer the mince tissue to a 15 milliliter sterile tube. Bring the volume to 4.5 milliliters with cold dissection medium and add 500 microliters of 2.5%trips into the tissue. Following this.
Wrap the tube in para film and place it in a 36 degree Celsius water bath for 15 minutes. In the meantime, mix it by inverting the tube every five minutes. Subsequently, transfer the tissue to a new 50 milliliter tube and try to minimize the tripsin containing dissection medium being transferred to the tube.
Bring the volume to 4.8 milliliters with fresh dissection medium, and add 200 microliters of D Ns to a final concentration of 60 micrograms per milliliter. Then triturate roughly 10 times with a sterile five or two milliliter pipette. Repeat this step as necessary.
Using a fire polished pasta pipette to dissociate most of the tissue, allow the remaining pieces of tissue to settle at the bottom for four to five minutes. Afterwards, remove the upper single cell suspension while leaving behind the settled pieces of tissue. Transfer it to a new tube with an equal volume of neuron plating medium and mix them gently.
Then count the cells using a hemo cytometer and plate them either on 15 millimeter cover slips as about 35, 000 cells in 200 microliters or on 60 millimeter dishes as about 1 million cells in three milliliters. Incubate the cells at 36.5 degrees Celsius for three to four hours after incubation. Combine the neuron layer and the secondary ggl for neurons on 60 millimeter dishes.
Wash the cells with warm DMEM. Change the medium to four milliliters of N two medium and add one thermon with ggl to each plate. With the GL facing the neurons for neuronal cover slips, transfer three to five cover slips to each 60 millimeter dish of secondary ggl with the neurons facing down 24 hours after plating the neurons add 10 micromolar of cytosine Beta D Arab azi to each dish to prevent glia proliferation shown here as an example of neurons from the neuroglia by laminar co-culture.
Five hours after plating processes are continuing to elongate at this stage. Here is an example of neurons from the neuroglia bi lanar co-culture 12 days after plating. At this stage, the extended axons and nites of the cells are clearly visible.
As neurons mature, the dendritic ABAs become more elaborate and develop synaptic contacts roughly two to three weeks after plating. Many dendritic spines are visible as shown in this figure. While performing this technique, it's important to work quickly and to maintain the sterility of each reagent is a lack of antibiotics in our culture.
Media makes the cells very susceptible to contamination.
