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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of the cerebellar system: in relation to its evolution, structure, and functions. , (1997).</li><li>Hatten, M. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Annual Review of Neuroscience. 18, 385-285 (1995).</li><li>Hatten, M. E., Gao, W. -. Q., Morrison, M. E., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in Culturing Nerve Cells. , 419-419 (1998).</li><li>Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of Cell Biology. 100 (2), 384-384 (1985).</li><li>Carletti, B., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroscientist. 14 (1), 91-91 (2008).</li><li>Knoepfler, P. S., Kenney, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Cycle. 5 (1), 47-47 (2006).</li><li>Manzini, M. C., Joseph, D. J., MacDermott, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular and Cellular Neuroscience. 35 (2), 328-328 (2007).</li><li>Manzini, M. C., Ward, M. S., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Journal of Neuroscience. 26 (22), 6040-6040 (2006).</li><li>Baptista, C. A., Hatten, M. E., Blazeski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 12 (2), 243-243 (1994).</li><li>Palay, S. L., Chan-Palay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cerebellar Cortex: Cytology and Organization. , (1974).</li><li>Messer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brain Research. 130 (1), 1-1 (1977).</li><li>Ruiz I Altaba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development. 126, 3205-3205 (1999).</li><li>Wallace, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Biology. 9 (8), 445-445 (1999).</li><li>Wechsler-Reya, R. J., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 22 (1), 103-103 (1999).</li><li>Solecki, D. J., Liu, X. L., Tomoda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 31 (4), 557-557 (2001).</li><li>Powell, S. K., Williams, C. C., Nomizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Journal of Neuroscience Research. 54 (2), 233-233 (1998).</li><li>Lander, A. D., Fujii, D. K., Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular and Cellular Biology. 20 (23), 9055-9055 (2000).</li><li>Kenney, A. M., Cole, M. D., Rowitch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development. 130 (1), 15-15 (2003).</li><li>Pons, S., Trejo, J. L., Martinez-Morales, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 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 ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell strainer with 70&mu;m mesh pore</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>Spinal Needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>405182</td>
<td>20G x 3-1/2 inches</td>
</tr>
<tr>
<td>Poly-D-Lysine mol wt>300,000</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1024</td>
<td>Each new batch of Poly-D-Lysine must be tested. 
</td>
</tr>
<tr>
<td>Percoll</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-1644</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin (100X)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4333</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14175-103</td>
<td>Must be calcium and magnesium free.</td>
</tr>
<tr>
<td>Neurobasal A-Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10888-022</td>
<td>&nbsp;</td>
<tr>
<td>Glutamax I supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum Free Supplement (50x)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>12-mm circle coverslips </td>
<td>&nbsp;</td>
<td>Carolina Biological Supply</td>
<td>63-3029</td>
<td>These are made in Germany by Deckgluder.</td>
</tr>
<tr>
<td>25-mm circle coverslips</td>
<td>&nbsp;</td>
<td>Carolina Biological Supply</td>
<td>63-3037</td>
<td>These are made in Germany by Deckgluder.</td>
</tr>
<tr>
<td>Papain Dissociation System </td>
<td>&nbsp;</td>
<td>Worthington Biochemical Corporation</td>
<td>LK 003150</td>
<td>Kit is good for five isolations.</td>
</tr>
<tr>
<td>Permaset scissors</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS6782</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 (Dumostar)</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS4978</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture dish</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture dish</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140685</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons at JoVE.com

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

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

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 ...

isolation-culture-post-natal-mouse-cerebellar-granule-neuron

Research

JoVE Journal

Biology

28.5K Aufrufe.  Columbia University. Hier präsentieren wir eine Methode zur Isolierung und Kultur des Kleinhirns Granulat Neuron Vorläuferzellen und Kleinhirn Nervenzellen Granulat aus postnatalen Maus.

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

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...