Name: Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons (Video) | JoVE Journal
Uploaded: 2009-01-16T00:00:00.000+00:00
Duration: 16 min 4 s
Description: Learn to isolate and culture cerebellar granule neuron progenitor cells from postnatal mice using papain dissociation and percoll gradient separation. This method enables neuroscience research.

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5 minutes

We thank Barbara Carletti and Anna Marie Kenney for invaluable suggestions. HYL is supported by the Training Grant &ldquo;Hormones: Biochemistry and Molecular Biology&rdquo;, DK07328. Supported in part by NIH grant 5R01 NS16951 (CAM).

Part 1: Setting up (1-2 days before dissection) (Not shown on video)
<ol>
<li>PREPARING CULTURE SOLUTIONS AND MEDIA:
<ul>
<li>4X CMF-PBS-EDTA (calcium and magnesium free-phosphate buffered saline-EDTA for Percoll dilutions): per liter, add 32 g NaCl, 1.2 g KCl, 8 g glucose, 2 g NaH2PO4, 1 g KH2PO4, 8 ml 2% NaHCO3 stock, 10 ml 1M EDTA (pH 8.0) to distilled/deionized water. Adjust the volume to 1 liter and the pH to 7.4. Filter sterilize.</li>
<li>HBSS-GLUCOSE: Add glucose (6 grams/liter) to calcium and magnesium free Hank&rsquo;s Balance Salt Solution (HBSS) and sterile filter. 500 ml can be stored at 4&deg;C for up to a month.</li>
<li>CULTURE MEDIA: serum-free medium (SFM) and 10% FBS medium. To 48 ml of Neurobasal A-Medium add 500 &mu;l 100X GlutaMAX I, 500 &mu;l 100X Penicillin-Streptomycin (final 100 Units penicillin and 100 &mu;g streptomycin), 6.25 &mu;l 2 M KCl (final 250 &mu;M). Split in 2 aliquots of 9 ml and 40 ml. To prepare 10% FBS medium, add 1ml of heat-inactivated FBS to the 9 ml aliquot. To prepare SFM, add 800 &mu;l of the serum-free supplement B-27 to the 40 ml aliquot. Filter sterilize and store at 4&deg;C for a maximum of 2 weeks. For best results make fresh media for each experiment.</li>
</ul>
</li>
<li>PREPARING PERCOLL DILUTIONS :
<ul>
<li>Percoll is supplied as a liquid and it must be acidified before use. To prepare the stock, add 1N HCl little by little to the Percoll solution over a 1-2 hour period until pH 7.4 is reached. Adding the HCl too rapidly will cause the Percoll to aggregate. Approximately 6.5ml of 1N HCl are needed for each liter of Percoll. Filter sterilize and store at 4&deg;C.</li>
<li>A 35% and a 60% (vol:vol) Percoll dilution will be used in the Percoll gradient step. Prepare 100 ml Percoll dilutions containing 35 or 60 ml of Percoll stock, 25 ml of 4X CMF-PBS-EDTA and distilled/deionized H2O to the final volume. Add a few drops of toluidine blue to the 60% solution; it will help visualize the interface and the cells. Filter sterilize and store at 4&deg;C.</li>
<li>4X CMF-PBS-EDTA should not be older than 3 months, as inadequate buffering will cause increased cell death during the procedure.</li>
</ul>
</li>
<li>PREPARING COVERSLIPS: Glass coverslips (best if from Carolina Biological Supply Company) are washed with 10% HCl overnight at room temperature with mild agitation, rinsed thoroughly with distilled/deonized water and stored in 70% ethanol. Before use, single coverslips are flame-sterilized by briefly holding them with forceps over an open flame (Bunsen burner) until the ethanol evaporates. 12-mm glass coverslips can be placed in 4-well culture plates and 25-mm coverslips in 6-well culture plates.</li>
<li>COATING CULTURE VESSELS WITH SUBSTRATE: For immunofluorescence staining or drug treatments glass coverslips are more appropriate since they can be easily mounted on glass slides for visualization under a microscope. When large numbers of cells are needed for collecting protein or RNA samples, cells can be cultured on plastic culture dishes.The night before dissection, glass coverslips or plastic dishes for culture must be coated with poly-D-lysine (500 &mu;g/ml; 800 &mu;l/well for 6-well plates or 350 &mu;l/well for 4-well plates). 5 mg/ml stock solution of poly-D-lysine is stored at -20&deg;C, then diluted in sterile distilled/deonized water and sterile filtered right before use. Culture vessels are incubated at 37&deg;C overnight (or for at least for 2 hrs before plating) and washed twice with sterile distilled/deonized water right before plating.</li>
<li>PREPARING PREPLATING DISHES: These dishes will be used for pre-plating the isolated cerebellar cells before culture to remove glial contaminants, which adhere more readily to a lower concentration of substrate. Coat two 60-mm plastic culture dishes with 4 ml per dish of poly-D-lysine (100 &mu;g/ml; Note this is 1/5 of the concentration used for culturing). Incubate at 37&deg;C overnight (or for at least for 2 hrs before dissection). Right before the dissection, remove the poly-D-lysine solution, wash the dishes twice with sterile water and allow to dry in the hood.</li>
<li>Sterilize dissection tools in the autoclave or on the day of dissection, immerse the tools in 70% ethanol for 20 minutes. Required tools: Permaset scissors, microdissecting scissors, four Dumont #5 dissecting forceps.</li>
</ol>
Part 2: Preparing for Dissection (day of dissection) &ndash; (Demonstrated in video)
All of the following procedures are performed in a tissue culture hood unless noted.
<ol>
<li>Wipe down the dissecting area with 70% ethanol. Warm the media to 37&deg;C in a water bath or in a 5% CO2 37&deg;C incubator.</li>
<li>When starting a new Papain Dissociation System Kit (Worthington), prepare the solution of albumin-ovomucoid inhibitor. Add 32 ml of EBSS (Earle&rsquo;s Balanced Salt Solution; provided in the kit) to the albumin-ovomucoid inhibitor mixture and allow the contents to dissolve while preparing the other components. Reconstitute for the first use, then store the remaining solution at 2-8&deg;C and use until the five isolations allowed by each kit are completed. </li>
<li>Add 5 ml of EBSS to one papain vial from the Dissociation Kit (each vial is sufficient for dissociation of 4 to 15 cerebella from postnatal day 5 mice). Place the papain vial in a 5% CO2 37&deg;C incubator or 37&deg;C water bath for 5 to 10 minutes until the papain is completely dissolved and the solution appears clear. Maintain the solution at room temperature during dissection.</li>
<li>Add 500 &mu;l of EBSS to one DNase vial from the Dissociation Kit. Mix gently by tapping the vial since DNase is sensitive to shear denaturation. Add 250 &mu;l of this solution to the vial containing the papain (final concentration is 20 units/ml papain and 0.005% DNase). Save the remaining DNase vial for use in step 5.1 or 6.1.</li>
</ol>
Part 3: Dissection and Meninges Removal
<ol>
<li>The optimal age for granule cell culture from C57BL/6J mice is postnatal day 4-6, when the number of granule cell progenitors in the EGL peaks1,10. Dissect pups one at a time and as you become more familiar with the cerebellar dissection try to reduce dissection time to no more than 6 minutes per pup. Speed is of the essence. 
If the Percoll gradient step is bypassed, eight postnatal-day-5 mouse pups can yield enough cells for one 6-well culture plate (or six 25-mm coverslips) or for six 4-well culture plates (or twenty-four 12-mm coverslips). Approximate yield is 4 to 5x106 cells per pup and plating densities can range between 1 and 5X105 cells/cm2. Since 15-20% cells are lost during the Percoll step4, more animals are needed to obtain the same desired cell density.</li>
<li>Pipette 15 ml of HBSS-glucose into a 60-mm plastic tissue culture dish and 10 ml into a sterile Falcon 15 ml conical plastic tube. Put on ice.</li>
<li>Outside the hood, wipe the head of the pup with 70% ethanol. Use Permaset scissors to decapitate the pup. (Decapitation will not be shown in the video.)</li>
<li>In the hood, hold the head with Dumont forceps so that you can clearly see the back of the skull. Access the brain by inserting microdissecting scissors into the foramen magnum and cutting straight toward the eyes. Using forceps, peel away the skin and lift up the skull to expose the brain. Using Dumont forceps, pinch off the cerebellum and the surrounding midbrain and transfer it to the dish with HBSS-glucose. (It is easier to manipulate the cerebellum and remove the meninges if the cerebellum is still attached to the surrounding tissue).</li>
<li>Under the dissecting microscope, gently peel the meninges off the cerebellum and also between the lobes with one fine Dumont #5 forceps while using the other forceps to anchor down the cerebellum to the plate. You will notice blood vessels on the surface of the cerebellum. These blood vessels are a good way to identify the meninges. Remove the meninges until the cerebellum takes on a matted white appearance. Separate the cerebellum from the rest of the midbrain. Turn it to its ventral side and remove the choroid plexus, which looks like a reddish ribbon between the ventral cerebellum and adjacent midbrain. Place each cerebellum in cold HBSS-glucose in a 15 ml conical tube on ice as soon as dissected. Change the dissecting solution to fresh HBSS-glucose after dissecting 3 brains.</li>
</ol>
Part 4: Cell suspension
<ol>
<li>Once all the dissections are finished, remove the HBSS-glucose from the tube and replace it with the papain solution made in step 2.4. Although the kit recommends cutting the tissue in small pieces, we find that it is not necessary to cut or mince the cerebella at this age. Place the tissue + papain solution (in a 15 ml conical tube) in a 37&deg;C water bath or 5% CO2 37&deg;C incubator for 15 to 20 minutes. For 4 to 8 cerebella, 15 minutes at 37&deg;C is sufficient; for a larger number of cerebella 20 to 30 minutes is better. Gently agitate the tube every 3 to 4 minutes.</li>
<li>Triturate the mixture with a sterile p1000 aerosol pipette tip that has been coated with FBS. Do this step gently to prevent any air bubble formation. Fire polished glass pipettes can be used, but we find that serum coated sterile p1000 aerosol tips work just as well. To coat the pipette tip with FBS, pipette FBS up and down twice just before use. About 10 up and down movements with the pipette are enough to dissociate the tissue. The solution will become cloudy. Allow the suspension to sit for 30-60 seconds so that any large pieces of undissociated tissue will settle to the bottom of the tube.</li>
<li>Remove the suspended cells (careful not to remove the undissociated tissue pieces at the bottom) into a fresh 15 ml conical tube with a serum coated pipette tip. Centrifuge at approximately 200xg for 5 minutes at room temperature. Prepare re-suspension medium. To do this, mix 2.7 ml EBSS with 300 &mu;l of reconstituted albumin-ovomucoid inhibitor solution in a sterile tube. Add 150 &mu;l of DNase solution from step 2.4.</li>
<li>After centrifuging, discard the supernatant and immediately resuspend the cell pellet in the diluted DNase/albumin inhibitor solution prepared in step 4.3 with a serum coated p1000 aerosol pipette tip.</li>
<li>Prepare a discontinuous density gradient as follows. Add 5 ml of albumin-ovomucoid inhibitor solution to a 15 ml conical tube. Carefully layer the cell suspension on top. Centrifuge at approximately 70xg for 6 minutes at room temperature. Dissociated cells pellet at the bottom of the tube, membrane fragments remain at the interface.</li>
<li>If you wish to obtain cultures that are enriched in granule cells, bypass the Percoll gradient separation step (which yields purer cultures of cerebellar granule cells), and proceed to Part 6. If you wish to further isolate the granule cells from glia and large interneurons by Percoll gradient separation, proceed to Part 5. Use of the Percoll gradient step will reduce the yield of cells. In our hands, the reduction in yield is approximately 20-35%. However, there is an increase in the enrichment of granule neurons and progenitor cells from approximately 90% to 95-99%.</li>
</ol>
Part 5: Percoll Gradient Separation
<ol>
<li>Discard the supernatant and immediately resuspend the pellet in 2 ml of HBSS-glucose with 50 &mu;l of DNase from step 2.4. Filter the cell suspension though a nylon mesh (pore size 70 &mu;m) by gravity. This step will remove large non-neuronal cells and provides for a better single cell suspension.</li>
<li>Prepare two fire-polished glass Pasteur pipettes. To do this, gently flame the tip of a glass pipette until the edge is smoothened and the pipette bore is 40-50% of the original size. Take care not to make the opening too small.</li>
<li>Prepare Percoll gradient. Place 10 ml of 35% Percoll solution in a 50 ml sterile conical tube. 60% Percoll (10 ml) is loaded into a 10 ml sterile syringe with a sterile spinal needle attached. Gently layer the 60% Percoll solution underneath the 35% solution, by adding the 60% solution at the bottom of the tube with the spinal needle. Take care to keep a sharp interface between the two layers. Add the cells to the top of the gradient using a fire-polished pipette, gently adding them along the side of the tube without disturbing the interface.</li>
<li>Carefully place the tube in the centrifuge and centrifuge at 1800xg. Ramp up the speed one step every 20 seconds (increments of speed are as follows: approximately 18, 70, 200, 440, 850, 1100, 1800xg) so that it takes approximately 2 minutes to reach the desired speed. Start the timer to 10 min when 1800xg is reached. When finished, decrease speed gradually one step every 20 seconds.</li>
<li>Remove the upper interface of the gradient (containing large glia, Purkinje cells, and interneurons) and discard.</li>
<li>Carefully remove the cells at the interface between the 35% and 60% Percoll with a fire-polished pipette and transfer to a 50 ml conical tube. Resuspend in 3 volumes of HBSS-glucose and mix by inverting several times. Centrifuge for 5 minutes at 1100xg.</li>
<li>Remove the supernatant and add 4 ml of 10% FBS medium with 50 &mu;l of DNase. Resuspend the pellet gently using a fire-polished pipette to form a single cell suspension. Proceed to step 6.2.</li>
</ol>
Part 6: Pre-plating and Plating
<ol>
<li>Discard the supernatant and immediately resuspend the pellet in 4 ml of 10% FBS medium with 50 &mu;l of DNase from step 2.4. Filter the cell suspension though a nylon mesh (pore size 70 &mu;m) by gravity. This step will remove large non-neuronal cells and provides for a better single cell suspension.</li>
<li>Plate the collected cells on a poly-D-lysine coated pre-plating dish for 20 minutes in a 5% CO2 37&deg;C incubator. Repeat with a fresh dish. The astroglia and heavier cells will settle down and adhere to the dishes and leave the small granule neurons and neuron progenitor cells floating. After the incubation, loosely adhered granule neurons and neuron progenitor cells are easily dislodged and loosened with gentle tapping of the plate.</li>
<li>Collect the granule neurons and neuron progenitor cells in a 15 ml conical tube and centrifuge at 200xg for 5 minutes. Resuspend the pellet in 1 ml of serum-free medium and count an aliquot of 10 &mu;l using a hemocytometer.</li>
<li>Add serum-free medium to reach the desired cell density. Rough guidelines to numbers of cells to plate for medium density: for 6-well plates, 3.5 to 4 million cells; for 4-well plates, 650,000 cells. For 6-well culture plates, I plate 1.5 ml /well and for 4 well-plates, I plate 0.5 ml/well. The cells are maintained in a 5% CO2 37&deg;C incubator. Change the medium completely 24 hours later with subsequent changes every two to three days.</li>
</ol>

<ol><li>Altman, J., Bayer, S. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAltman%2C+J+%22Development+of+the+cerebellar+system%3A+in+relation+to+its+evolution%2C+structure%2C+and+functions%22">Development of the cerebellar system: in relation to its evolution, structure, and functions</a>. , CRC Press. Boca Raton. (1997).</li>
<li>Hatten, M. E., Heintz, N. Annual Review of Neuroscience. 18, 385-285 (1995).</li>
<li>Hatten, M. E., Gao, W. -Q., Morrison, M. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHatten%2C+ME+%22in+Culturing+Nerve+Cells%22">in Culturing Nerve Cells</a>. Banker, G., Goslin, K. , MIT Press. Cambridge, Mass. 419-419 (1998).</li>
<li>Hatten, M. E. The Journal of Cell Biology. 100 (2), 384-384 (1985).</li>
<li>Carletti, B., Rossi, F. Neuroscientist. 14 (1), 91-91 (2008).</li>
<li>Knoepfler, P. S., Kenney, A. M. Cell Cycle. 5 (1), 47-47 (2006).</li>
<li>Manzini, M. C., Joseph, D. J., MacDermott, A. B. Molecular and Cellular Neuroscience. 35 (2), 328-328 (2007).</li>
<li>Manzini, M. C., Ward, M. S., Zhang, Q. Journal of Neuroscience. 26 (22), 6040-6040 (2006).</li>
<li>Baptista, C. A., Hatten, M. E., Blazeski, R. Neuron. 12 (2), 243-243 (1994).</li>
<li>Palay, S. L., Chan-Palay, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPalay%2C+SL+%22Cerebellar+Cortex%3A+Cytology+and+Organization%22">Cerebellar Cortex: Cytology and Organization</a>. , Springer. Berlin, Heidelberg, New York. (1974).</li>
<li>Messer, A. Brain Research. 130 (1), 1-1 (1977).</li>
<li>Ruiz I Altaba, A. Development. 126, 3205-3205 (1999).</li>
<li>Wallace, V. A. Current Biology. 9 (8), 445-445 (1999).</li>
<li>Wechsler-Reya, R. J., Scott, M. P. Neuron. 22 (1), 103-103 (1999).</li>
<li>Solecki, D. J., Liu, X. L., Tomoda, T. Neuron. 31 (4), 557-557 (2001).</li>
<li>Powell, S. K., Williams, C. C., Nomizu, M. Journal of Neuroscience Research. 54 (2), 233-233 (1998).</li>
<li>Lander, A. D., Fujii, D. K., Reichardt, L. F. Proceedings of the National Academy of Sciences of the United States of America. 82 (7), 2183-2183 (1985).</li>
<li>Kenney, A. M., Rowitch, D. H. Molecular and Cellular Biology. 20 (23), 9055-9055 (2000).</li>
<li>Kenney, A. M., Cole, M. D., Rowitch, D. H. Development. 130 (1), 15-15 (2003).</li>
<li>Pons, S., Trejo, J. L., Martinez-Morales, J. R. Development. 128, 1481-1481 (2001).</li>
</ol>

This protocol is based on modifications of procedures that have been described in the past3,7,11. There are several important points to note as discussed below.
The granule neurons and progenitor cells adhere within 2 hours of plating. Healthy cells have a round morphology under the phase-contrast microscope4. Within 24 hours after plating, healthy cells will spread evenly around the coverslips or the plastic well and will form processes. At the time of the first medium ...

<table><tbody><tr><td>Cell strainer with 70&amp;mu;m mesh pore</td><td>BD Biosciences</td><td>352350</td><td></td></tr><tr><td>Spinal Needle</td><td>BD Biosciences</td><td>405182</td><td>20G x 3-1/2 inches</td></tr><tr><td>Poly-D-Lysine mol wt&amp;gt;300,000</td><td>Sigma-Aldrich</td><td>P1024</td><td>Each new batch of Poly-D-Lysine must be tested. </td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P-1644</td><td></td></tr><tr><td>Penicillin-Streptomycin (100X)</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14175-103</td><td>Must be calcium and magnesium free.</td></tr><tr><td>Neurobasal A-Medium</td><td>Invitrogen</td><td>10888-022</td><td></td></tr><tr><td>Glutamax I supplement</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>B-27 Serum Free Supplement (50x)</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>12-mm circle coverslips </td><td>Carolina Biological</td><td>63-3029</td><td>These are made in Germany by Deckgluder.</td></tr><tr><td>25-mm circle coverslips</td><td>Carolina Biological</td><td>63-3037</td><td>These are made in Germany by Deckgluder.</td></tr><tr><td>Papain Dissociation System </td><td>Worthington Biochemical</td><td>LK 003150</td><td>Kit is good for five isolations.</td></tr><tr><td>Permaset scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS6782</td><td></td></tr><tr><td>Dumont #5 (Dumostar)</td><td>Roboz Surgical Instruments Co.</td><td>RS4978</td><td></td></tr><tr><td>4-well culture dish</td><td>Nalge Nunc international</td><td>176740</td><td></td></tr><tr><td>6-well culture dish</td><td>Nalge Nunc international</td><td>140685</td><td></td></tr></tbody></table>

isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons 

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

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.

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JoVE Journal

Biology

This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

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Spinal Cord Neurons Isolation and Culture from Neonatal Mice

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum

Learn to isolate and culture cerebellar granule neuron progenitor cells from postnatal mice using papain dissociation and percoll gradient separation. This method enables neuroscience research.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

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