Name: isolation and purification of drosophila peripheral neurons by magnetic bead sorting
Uploaded: 2009-12-01T14:59:00
Description: Watch this Scientific Journal Video about Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting at JoVE.com

We thank Drs. Yuh-Nung Jan and Wes Grueber for providing fly stocks used in this study. The authors acknowledge the Thomas F. and Kate Miller Jeffress Memorial Trust for support of this research (D.N.C.) and the George Mason University Provost&#x2019;s Office (E.P.R.I.).

General Comments on Magnetic Bead Sorting of Drosophila Peripheral Neurons (Total Timing for the Completion of the Protocol: 2.5-3 hours)
Standard lab procedures for maintaining a clean, RNAse free environment must be observed at all times to prevent RNA degradation.
When the Drosophila larval cuticle is dissected and placed in the cell dissociation buffer, the peripheral neurons are one of the last cells to detach from the cuticle. We have exploit...

     

<ol>
	<li>Corty, M. M., Matthews, B. J., Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecules+and+mechanisms+of+dendrite+development+in+Drosophila.">Molecules and mechanisms of dendrite development in Drosophila.</a> Development. 136, 1049-1061 (2009).</li><li>Parrish, J. Z., Emoto, K., Kim, ., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+that+regulate+establishment,+maintenance,+and+remodeling+of+dendritic+fields.">Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields.</a> Ann. Rev. Neurosci. 30, 399-423 (2007).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Song, W., Onishi, M., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+multidendritic+sensory+neurons+are+necessary+for+rhythmic+locomotion+behavior+in+Drosophila+larvae.">Peripheral multidendritic sensory neurons are necessary for rhythmic locomotion behavior in Drosophila larvae.</a> Proc Natl Acad Sci USA. 104, 5199-5204 (2007).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+methods.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) methods.</a> Methods. 25, 402-408 (2001).</li></ol>

The protocol presented here is optimized for the isolation and purification of peripheral neurons which adhere tightly to the inner surface of the Drosophila third instar larval cuticle using a magnetic bead cell sorting strategy. While we have used this protocol to specifically isolate Drosophila da neurons, applications of this protocol to the isolation of other cell types that adhere to the cuticle in larval or pupal stages of development (e.g. epithelia, muscle, other peripheral neurons) can be adapted by varying a ...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<td>10X Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>MP Bioproducts</td>
<td>PBS10X02</td>
<td>Diluted to 1X working solution</td>
</tr>
<tr>
<td>10X Liberase Blendzyme 3</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11814176001</td>
<td>Diluted to 1X working solution (28 W&uuml;nsch units/vial)</td>
</tr>
<tr>
<td>RNase-AWAY</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>83931</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated Rat anti-Mouse-CD8a antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>MCD0815</td>
<td>100 &mu;g/ml stock concentration</td>
</tr>
<tr>
<td>BSA (Bovine Serum Albumin), Fraction V</td>
<td>&nbsp;</td>
<td>GibcoBRL</td>
<td>11018-017</td>
<td>Prepare a 1% BSA solution in PBS</td>
</tr>
<tr>
<td>Dynabeads M-280 Streptavidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11205D </td>
<td>1 &mu;l can bind 0.05-0.10 &mu;g of biotinylated antibody</td>
</tr>
<tr>
<td>PicoPure RNA Isolation Kit</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>KIT0204</td>
<td>Follow manufacturer&#x2019;s instructions</td>
</tr>		</tbody>
		</table>
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			Equipment
<ul>
 <li>(10) Neodymium block magnets (K &amp; J Magnetics, Inc., Cat. #B444B), alternatively DynaMag&trade;-2 (Invitrogen, Cat. #123-21D) may be used. </li>
 <li>Kontes Glass Tissue Grinder, 2 ml working capacity, with large clearance pestle (Cat. #885300-0002)</li>
 <li>Cell filters, e.g. MACS Pre-Separation Filter (Miltenyi Biotec, Cat. #130-041-407)</li>
 <li>35 mm petri-dishes coated with Sylgard (Dow Corning Corporation, Cat. #3097358-1004)</li>
 <li>Dissecting tools: two pairs of Dumont No. 5 forceps (Fine Science Tools, Cat. #11251-20)</li>
 <li>Vannas spring micro-dissection scissors (Fine Science Tools, Cat. #15000-08)</li>
 <li>Pipettes (P-1000, P-100, P-10) with disposable tips</li>
 <li>Standard hemocytometer to count the cells and estimate purity</li>
 <li>Pasteur pipettes with fire polished tips of standard diameter, narrowed to ~50% of standard diameter and narrowed to ~25% of standard diameter for trituration</li>
 <li>Table top micro-centrifuge (1-16,000 (x) g)</li>
 <li>Vortex</li>
 <li>Fluorescent stereo-microscope (a Leica MZ16FA was used in this protocol)</li>
</ul>		</div>

isolation and purification of drosophila peripheral neurons by magnetic bead sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Watch this Scientific Journal Video about Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting at JoVE.com

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

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