This procedure begins with removing the cerebellum from P four to P five. Newborn mice meninges and the choroid plexus are removed from individual cerebella, which are then collected in a 15 milliliter conical tube. The cerebella are dissociated with papain and brought to a single cell suspension with gentle tation.
This suspension is filtered by gravity through a nylon mesh with a 70 micron pore size. The flow through is gently layered on top of a discontinuous perca gradient and centrifuged to separate the granule cells from the glia into neurons and Perkin gene neurons. Cells in the interface of the gradient are collected and prepl on poly de lycine coated plates.
To further remove the glial cells, granule cells are collected, counted and plated at desired densities on poly de lycine coated glass cover slips or plastic culture plates. Hi, I am hey young Lee from The laboratory of Dr.Lloyd Green in the Department of Pathology at Columbia University. Today we'll show you a procedure for isolating and culturing cerebellar granule neurons from postnatal mice.
We use this procedure in our laboratory to study granule neuro progenitor proliferation and differentiation. So let's get started. This procedure begins with using 70%ethanol in the dissection area to dissociate cerebellar granule cells, we use the Papin dissociation system.
When starting a new kit, prepare the albumin OVO equate MOID inhibitor solution. To do this, take 32 milliliters of Earl's balanced salt solution or EBSS, which is provided in the kit, and add it to the albumin OVO moid mixture. Set it aside while preparing the other components to allow the contents to dissolve.
Next, add five milliliters of EBSS to one papain vial from the dissociation kit. Each vial is sufficient to dissociate four to 15 cerebella from P five mice. Then place the papain vial in a 5%CO 2 37 degrees Celsius incubator for five to 10 minutes until the papa is completely dissolved and the solution appears clear.
Maintain the solution at room temperature during dissection. Next, add 500 microliters of EBSS to 1D NAS vial from the kit. Mix gently by tapping the vial.
Since DNAS is sensitive to sheer denaturation, add 250 microliters of this solution to the vial of pepane to obtain a final concentration of 20 units per mil and 0.005%DNA, save the remaining DNA file for use in later steps. Once the tools and reagents are ready, proceed with dissecting the cerebella and removing the meninges for this procedure. C 57 black six mice are used between postnatal days four and six.
This age range is optimal for granule cell culture because the number of external granule layer or EGL granule cell progenitors is at a maximum to begin pipette 15 milliliters of HBSS glucose into a 60 millimeter plastic tissue culture dish and 10 milliliters into a sterile 15 milliliter conical tube. Place the tube on ice, wipe the head of the pup with 70%ethanol. Use perce scissors to remove the head.
Transfer the head into the hood and hold it with forceps so the back of the skull is clearly visible. Insert the micro dissecting scissors into the foremen magnum in order to access the brain and then cut straight toward the eyes. Use fine dumont.
Number five, forceps to peel away the skin and lift up the skull to expose the brain. Then pinch off the cerebellum and the surrounding midbrain. Transfer it to the dish containing HBSS glucose.
Note that it is easier to manipulate the cerebellum and to remove the meninges if the cerebellum is still attached to the surrounding tissue. Next, place the tissue under a dissecting microscope. Then use one set of forceps to anchor down the cerebellum to the plate.
Use fine dumont. Number five, forceps to gently peel the meninges off the cerebellum as well as between the lobes. You will notice blood vessels on the surface of the cerebellum.
These blood vessels are a good way to identify the meninges. Continue to remove the meninges until the cerebellum takes on a matted white appearance. Then separate the cerebellum from the rest of the midbrain.
Turn it on its ventral side, and remove the choroid plexus, which looks like a reddish ribbon between the ventral cerebellum and the adjacent midbrain. As soon as the cerebellum is dissected, place it in cold HBSS glucose in the 15 milliliter conical tube on ice. After dissecting three brains, replace the dissection solution with fresh HBSS glucose.
As you become more familiar with cerebellar dissection, try to reduce the dissection time to no more than six minutes per pup. When the dissections are complete, proceed with the cell suspension. Step to Generate a cell suspension from the cerebellar dissections.
First, remove the HBSS glucose from the tube and replace it with papain solution. Although the kit recommends cutting the tissue into small pieces, we find that it is not necessary at this age. Place the tissue and papain solution in a 5%CO 2 37 degree incubator for 15 to 20 minutes.
For a four to eight cerebella incubate for 15 minutes. For a larger number incubate for up to half an hour. Gently agitate the tube every three to four minutes.
Once the incubation is complete, begin the tation step coat a sterile P 1000 aerosol pipette tip with FBS by pipetting up and down twice. Now trigger rate the mixture. Make sure to pipette gently to prevent any air bubbles from forming.
About 10 up and down movements with the pipette are enough to dissociate the tissue. The solution should become cloudy. Allow the suspension to sit for 30 to 60 seconds so that any large pieces of an unassociated tissue will settle to the bottom of the tube.
Although fire polished glass pipettes can be used for tation, we find that serum coated sterile P 1000 tips work just as well. Next, remove the suspended cells into a fresh 15 mil conical tube using a serum coated pipette tip, be careful not to remove the unassociated tissue pieces at the bottom. Then centrifuge the cell suspension at approximately 200 G for five minutes.
At room temperature during the spin, prepare the resus suspension medium. To do this, add 2.7 milliliters of EBSS in a sterile tube. Then add 300 microliters of the reconstituted albumin OVO MOID inhibitor solution.
Finally, add 150 microliters of the DNA solution. Once the centrifugation is complete, discard the supernatant and immediately resuspend the cell pellet in the diluted DNA albumin inhibitor solution. Again, use a serum coated pipette tip to resuspend the pellet.
Now prepare a discontinuous density gradient. To do this, add five milliliters of the albumin oval MOID inhibitor solution to a 15 milliliter conical tube. Layer the cell suspension on top of the solution by gently adding the cells along the side of the tube, then centrifuge at approximately 70 G for six minutes.
At room temperature, the dissociated cells will pellet at the bottom of the tube while the membrane fragments remain at the interface. If you wish to obtain cultures that are enriched in granule cells, there is no need for further purification. Discard the SUP natant and immediately resuspend the pellet in four milliliters of 10%FBS medium with 50 microliters of DNAs.
Filter this suspension by gravity through a nylon mesh with a poor size of 70 microns. This step removes large non neuronal cells and provides for a better single cell suspension. Then bypass the following per call gradient separation step and proceed to the cell plating step.
Or if you wish to further purify the granule cells from glia and large inter neurons proceed to the per call gradient. Step, Discard the snat and immediately resus. Suspend the pellet in two milliliters of HBSS glucose with 50 microliters of DNAs.
Again, filter the suspension through a nylon mesh Before beginning the perca gradient separation, make two fire polished pasture pipettes. Gently flame the tip of a pipette until the edge is smooth and the pipette bore is about half of the original size. Be careful not to make the opening too small.
Now make the per call gradient. Place 10 mils of 35%per call solution into a 50 mil sterile conical tube. Then load 10 mils of 60%per call into a 10 mil sterile syringe with a sterile spinal needle attached.
Gently layer the 60%per call solution underneath the 35%solution by letting the syringe tip touch the bottom of the tube. Take care to keep a sharp interface between the two layers. Next, add the cells to the top of the gradient.
Using a fire polished pipette, gently add the cells along the side of the tube without disturbing the interface. After layering the cells carefully place the tube in the centrifuge. Start centrifuging at approximately 18 G.Ramp up the speed one step every 20 seconds so that it takes approximately two minutes to reach 1800 G.After 10 minutes at 1800 G, decrease the speed gradually one step every 20 seconds.
When the spin is complete, remove the upper interface of the gradient and discard it. This interface contains large ggl bikini cells and inter neurons. Next, use a fire polished pipette to carefully remove the cells at the interface.
Transfer the cells to a 50 mil tube. If the volume happens to be greater than 15 mils, then split them into two 50 mil tubes and resuspend in three volumes of HB S'S glucose and mix by inverting several times. Then centrifuge for five minutes at 1100 G.Now remove the supernatant and add four mils of medium containing 10%FBS and 50 microliters of D ns reus.
Suspend the pellet gently using a fire polished pipette to form a single cell suspension. Once the cells are resuspended, proceed with prepl. The cells prelate the collected cells on a poly de lysine coated dish for 20 minutes.
In an incubator at 37 degrees Celsius and 5%CO2. During the incubation, the astroglia and heavier cells will settle down and adhere to the dishes while the small granule neurons and neuron progenitor cells will remain floating or loosely attached. After 20 minutes, gently tap on the dish to dislodge loosely adhered granule cells.
Then transfer the cell suspension to a fresh poly de lycine coated plate and repeat the incubation. Next, collect the granule neurons and neuron progenitor cells in a 15 mil conical tube and centrifuge at 200 G for five minutes. Then resuspend the pellet in one mil of C free medium and count using a hemo cytometer.
Finally, add serum free medium to reach the desired cell density to plate at medium density. For six wall plates. Use approximately three and a half to 4 million cells for four wall plates.
Use 650, 000 cells. Maintain the cells in a 37 degree incubator with 5%CO2. Change the medium completely 24 hours after the initial plating, then every two to three days thereafter.
Here are some images of healthy granule neuron and progenitor cell cultures. 24 hours after plating and six days after plating. These cells can be maintained in culture for up to two weeks.
Note that within 24 hours after plating, healthy cells will spread evenly around the cover slips or the plastic well and will project processes. Cell clumping and the presence of varicosities on the cell processes indicate that the cells are unhealthy or that the poly de lycine substrate is toxic. As seen in the diagram, granule neuron progenitors begin to differentiate upon plating and express markers.
Representative of their differentiation. State the proliferation of granule neuron progenitors can be substantially prolonged by adding sonic hedgehog to the medium. Laminin can also be added as a substrate in addition to polylysine to promote neurite outgrowth.
These features of the granule cell culture offer a system for studying either the biology of granule neurons or the regulation of progenitor cell differentiation into neurons. We've just shown you how to Isolate and culture MAO cerebellar grand neuro progenitors and neurons. When doing this procedure, it's important to remember the approximate yield is 4 2 5 times 10th.
Of the six cells per pup and plating densities can range between and five times center the fifth cells per centimeter square. So if the perico gradient step is bypassed, then eight postnatal day five mouse pups can yield enough cells for six four well culture plates in my hands, the use of the perico gradient step will reduce the yield of cells approximately 2230 5%However, the percentage of granule neurons in cultures increases from 90 to 95 to 99%enrich granule cells obtained without the per cot gradient separation step have been used to study the regulation of gran neuron progenitor proliferation and differentiation. However, if the cultures are to be used beyond several days in vitro inclusion of the per cot gradient step is recommended in order to decrease the contamination of the gran neurons by proliferating non neuronal cells.
Also, make sure the solutions used for making the per cot gradient is fresh as improper buffering leads to greater cell death. So that's it. Thanks for watching and good luck with your experiments.
