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 exploited this property and designed this protocol to remove most of the non-specific cells from the cuticle such as muscle and fat prior to isolating the da neurons.
With practice, the whole protocol can be successfully completed within approximately 2.5 hours. The preparation of antibody coated beads must be completed before the experiment begins.
1. Preparing Magnetic Beads for Binding Cells:
This step must be completed prior to the start of the experiment. The prepared beads can be prepared and stored at 4&deg;C until needed.
<ol>
 <li>Wash 100 &mu;l of Dynabeads M-280 Streptavidin coated beads three times in PBS by resuspending it in 500 &mu;l of fresh PBS and pelleting it in a strong magnetic field each time.</li>
 <li>Finally resuspend the beads directly in 100 &mu;l of undiluted biotinylated rat anti-mouse-CD8a antibody (antibody concentration is 100 &mu;g/ml).</li>
 <li>Incubate the mixture for 1 hour on ice with occasional mild vortexing to prevent sedimentation. [1 &mu;l of Dynabeads M-280 can bind 0.05-0.10 &mu;g of biotinylated antibody].</li>
 <li>Wash the bead-antibody mixture three times as described in step 1.1 to remove the excess antibody. The magnetic beads are now coated with antibody and ready to be used. Store the bead-antibody mixture in 100 &mu;l of 1X PBS at 4&deg;C until use.</li>
</ol>
2. Selecting and Washing Larvae: (10-15 minutes)
 <ol>
 <li>Pick 30-50 age matched third instar larvae and place them in a 1.6ml microfuge tube with 1-1.2 ml of 1X Phosphate Buffered Saline (PBS). (3-5 minutes)</li>
 <li>Close the tube and vortex it at the maximum setting three times for 1 second each. </li>
 <li>Using a fire-polished Pasteur pipette discard the supernatant completely. Repeat the wash (2.1) and vortex (2.2) steps 3-4 times until the supernatant is visibly clear of any food particles and debris. </li>
 <li>Briefly repeat the wash with 1 ml of 70% ethanol and discard the supernatant. </li>
 <li>Wash the larvae twice with 1 ml of ddH2O and discard the supernatant.</li>
 <li>Briefly repeat the wash once again with 1 ml of RNase-AWAY and discard the supernatant.</li>
 <li>Wash the larvae three times in 1 ml of ddH2O to ensure complete removal of RNase-AWAY.</li>
</ol>
3. Dissection: (10-12 minutes)
<ol>
 <li>Place 10-12 larvae on the center of a Sylgard coated 35mm Petri plate. Position them slightly away from each other. [Critical Step: Discard any larvae that do not seem to be at the appropriate developmental stage]</li>
 <li>Cut open the anterior tip of the larvae using a pair of fine dissection scissors for all the larvae.</li>
 <li>Using a pair of dull Dumont No. 5 forceps invert the larvae inside-out. Insert one forcep inside the larval cuticle all the way to the posterior end. Pinch the tips of the forceps together to grab the posterior end of the cuticle (Figure 1a) (try pressing the cuticle down on the Sylgard surface to make it easier). Using the second pair of forceps, push the larval cuticle inside out. [Critical Step: Try practicing this method a few times before attempting the cell isolation experiment. Try to get the larvae completely inverted, to ensure that all the soft tissues are exposed to the solution for easy dissociation.]</li>
 <li>After dissecting 3-4 larvae, transfer them immediately to fresh, ice-cold PBS (place the tube on ice) in a 1.6 ml microfuge tube.</li>
 <li>Repeat steps 3.1 to 3.4 until all the required larvae are collected (30-40 larvae in this protocol). [Critical Step: Anticipate 10-20% loss during dissection and dissociation, and plan accordingly.]</li>
 </ol>
4. Removal of loosely adherent non-specific cells: (2-3 minutes)
 [This step aids the clearing of loosely adherent non-specific tissues such as fat bodies and CNS.]
<ol>
<li>Take the 1.6 ml microfuge tube containing the inverted larval cuticles and replace the supernatant with approximately 700-800 &mu;l of fresh ice-cold PBS.</li>
<li>Pulse-vortex the microfuge tube 5 times (3 seconds per pulse) at full speed.</li>
<li>Discard the supernatant and replace it with approximately 700-800 &mu;l fresh, ice-cold PBS.</li>
<li>Repeat steps 4.2 and 4.3 three times.</li>
<li>Resuspend the larval cuticles in 400 &mu;l of fresh, ice-cold PBS</li>
</ol>
5. Dissociating the tissue into a single cell suspension: (18-20 minutes)
[Critical Step: Over-dissociation may cause the loss of the cell-surface marker leading to poor cell yield and low cell viability. The larval tissue can be dissociated by either mechanical dissociation (sonication, douncing), enzymatic dissociation (trypsin, collagenase etc.) or a combination of both. As these larval tissues are difficult to dissociate, we found that a combination of both mechanical and enzymatic dissociation yielded the best results.]
<ol>
 <li>Add 1.5 &mu;l of 1X Liberase Blendzyme 3 (28 W nsch units/vial) to the larval cuticle suspended in 400 &mu;l of PBS.</li>
 <li>Vortex the solution 2-3 times for 1 second each at maximum setting (This should remove the loosely adherent cells from the cuticle into the solution).</li>
 <li>Incubate the solution at room temperature (22-25&deg;C) for 5 minutes. [Critical Step: Incubation time greatly affects the final cell sorting efficiency. The recommended incubation time should be sufficient for loosening the tissue. Do not exceed 15 minutes.]</li>
 <li>Pulse-vortex the tube 20-30 times at maximum settings for 2 seconds per pulse at full speed. This should releases the muscle and other tissue into the solution. Inspect a small sample from the solution under a fluorescent stereo-microscope at each step. (With experience one should be able to determine the level of dissociation by observing the microfuge tube directly under a fluorescence enabled stereo-microscope) </li>
 <li>Wash the larval cuticles 2-3 times with fresh, ice-cold PBS and finally resuspend them in 500 &mu;l of fresh, ice-cold PBS containing 1% BSA.</li>
 <li>To avoid the larval cuticle sticking to the glass surface of the 2 ml Kontes tissue grinder and large clearance pestle, pre-coat the tissue grinder and pestle with a 1% BSA in PBS solution and after a brief rinse discard the BSA solution. Subsequently, using a fire-polished Pasteur pipette, transfer the cuticles from step 5.5 to the BSA-coated tissue grinder. [Critical Step: Pre-cool the tissue grinder/pestle by placing it on ice for a few minutes to prevent cell damage/lysis]. </li>
 <li>Dounce the tissue with slow and steady strokes, avoiding frothing (approximately 20-30 strokes). [Critical Step: Dounce slowly and steadily, otherwise the cells may lyse.] </li>
 <li>To assess the cell dissociation levels, wipe the outer wall of the tissue grinder with a clean Kimwipe tissue, and inspect it under a fluorescent stereo-microscope. The neurons should have detached from the cuticle, and can be seen in the solution. If this is found to be difficult, alternatively pipette out a small sample of the solution, and observe it under a fluorescent stereo-microscope. A good indication of cell dissociation is the absence of neurons from the larval cuticle. However, if one still observes neurons attached to the cuticle, or observes incompletely dissociated cells, dounce further until the cells achieve a single cell suspension.</li>
 <li>Triturate the solution 5 times with a fire-polished Pasteur pipette narrowed to approximately 50% of the standard tip diameter, followed by 10 times with a fire-polished Pasteur pipette narrowed to approximately 25% of the standard tip diameter. [Critical Step: Forceful trituration may damage the cells. Monitor the cells between steps, and adjust the procedure accordingly]. </li>
 <li>Filter the solution through a 30 &mu;m cell filter and collect the cell filtrate in a 1.6 ml microfuge tube. The resulting solution should consist of a single cell suspension and is now ready for magnetic cell sorting.</li>
 </ol>
6. Magnetic Bead Cell Sorting: (45 - 75 minutes, dependent on antibody incubation time)
<ol>
<li>Add 15 &mu;l of antibody coated magnetic beads to 500 &mu;l of cell suspension (step 5.10). The remaining antibody conjugated magnetic beads can be stored until needed for subsequent cell isolations.</li>
 <li>Incubate the cells with antibody coated magnetic beads for 30-60 minutes on ice with occasional hand-mixing. [Critical Step: Incubation at a higher temperature or longer time may result in non-specific antibody binding.] </li>
 <li>Place the microfuge tube in a strong magnetic field for 2 minutes. All positively selected cells along with unbound beads will separate to the side of the tube. </li>
 <li>Slowly pipette the supernatant, making sure not to disturb the cell pellet. </li>
 <li>Wash the cells 3-4 times in fresh, ice-cold PBS to remove any remaining non-specific cells. </li>
 <li>Resuspend the cells in 30 &mu;l of fresh, ice-cold PBS.</li>
 <li>To approximate the purity and yield of cells, pipette 5 &mu;l of the cell suspension on the polished surface of a hemocytometer and count all the visible fluorescent cells under a fluorescent stereo-microscope. Also check the amount of non-fluorescent cells and any signs of impurities. Typically the sample will be highly enriched for fluorescent cells. </li>
 </ol>
7. RNA Isolation from Magnetic Bead Sorted Cells: (60 &#x2013; 75 minutes)
<ol>
 <li>After counting, pellet the cells in a magnetic field, discard the supernatant and add 20 &mu;l of extraction buffer from the PicoPure&trade; RNA Isolation Kit (Molecular Devices). Depending upon cell number one may need to add a higher volume of extraction buffer.</li>
 <li>Vortex the tube at maximum speed to enable the mixing of cell pellet with extraction buffer.</li>
 <li>Incubate the tube at 42&deg;C for 30 minutes. </li>
 <li>To ensure removal of the magnetic beads prior to column purification of the RNA (see step 7.5), the tube is briefly centrifuged at 2,000 (x) g for 2 minutes to pellet the magnetic beads. The tube is then placed in a strong magnetic field to retain the pellet, and the supernatant is transferred to a new microfuge tube. </li>
 <li>Extract and column purify the RNA according to the PicoPure RNA extraction kit manufacturer&#x2019;s instructions. DNAse treatment is optional, and can be performed on column during the RNA purification according to the analysis requirement. Finally, elute the bound total RNA in a small volume (11-30 &mu;l) of elution buffer and store at -80&deg;C until ready for use. If desired a 1 &mu;l aliquot may be used to assess total RNA quality on a Bioanalyzer 2100 (Agilent Technologies, Inc.).</li>
</ol>
Representative Results:
Magnetic bead sorting was used to isolate Drosophila da neurons (Figure 1). The RNA purified from these isolated da neurons (Figure 2a) was found to be of excellent quality as indicated by the presence of sharp 5.8S, 18S and 28S ribosomal RNA peaks when analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) (Figure 2b). Beginning with 30-40 third instar larvae we were capable of isolating on average 300-500 class-IV da neurons using the ppk-GAL4 driver, and 1500-2000 da neurons (Class I,II,III &amp; IV) using the pan-da neuron-specific GAL421-7 driver. To assess the neuronal-specific enrichment of our isolated cells we performed quantitative reverse transcription PCR (qRT-PCR) using two neuronal gene-specific markers (elav and futsch). These analyses revealed significant fold enrichment of both marker genes indicating a highly specific enrichment for da neurons as compared to flow through using our protocol (Figure 3). Finally, the isolated RNA from both pan-da neurons and class-IV da neurons was used to perform transcriptional expression profiling on Agilent Drosophila melanogaster whole-genome oligo microarrays (4 x 44K) (Figure 4). These analyses identified numerous previously implicated regulators of da neuron dendrite morphogenesis in addition to a broad spectrum of previously uncharacterized molecules and putative signal transduction pathways that potentially play important functional roles in da neuron development. Studies designed to assess the potential role(s) of these previously uncharacterized molecules in mediating da neuron development, and specifically dendrite morphogenesis, are presently underway.
<img src="/files/ftp_upload/1599/1599figure1tmb.jpg" border="0" /> Figure 1: Schematic of magnetic bead sorting of Drosophila da neurons. (a) Age matched third instar larvae bearing the da neuron-specific GAL4,UAS-mCD8-GFP reporter transgene are dissected by inverting the larval cuticle inside-out, to expose the PNS to dissociation buffer and stored in ice-cold PBS. (b) Enzymatic dissociation is carried out by adding Liberase Blendzyme 3 to the solution containing larval cuticle. (c) The larval tissues are further dissociated by a combination of vortexing, trituration and douncing to remove non-specifically labeled tissues such as fat-bodies, gut and CNS. (d,e) The cells are then filtered using a 30 &mu;m cell filter. The solution contains a single cell suspension of different cell types including epithelia, muscle and neurons. (f) Anti-mouse CD8a-antibody coated Dynabeads M-280 are added to the cell suspension, and incubated on ice for 30-60 minutes. (g) The magnetic beads binds to the da neurons that are expressing a mouse CD8 tagged GFP fusion protein. (h,i) The magnetic bead coated cells are separated by placing the solution in a strong magnetic field. The supernatant is discarded, and the cells are washed three times to remove any residual non-specific cells, resulting in (j) highly purified populations of da neurons. Please <a href="https://www.jove.com/files/ftp_upload/1599/1599figure1.jpg">click here</a> to see a larger version of figure 1.
<img src="/files/ftp_upload/1599/1599figure2tmb.jpg" border="0" /> Figure 2: (a) Representative image of positively selected, GFP fluorescent class-IV da neurons isolated by cell dissociation and magnetic bead sorting. The resulting population of neurons was determined to be highly enriched for class-IV da neurons with little or no contaminating cell impurities. (b) An Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) electropherogram of total RNA isolated from magnetic bead sorted da neurons, showing an excellent total RNA quality as indicated by the presence of 5.8S, 18S, and 28S rRNAs. Please <a href="https://www.jove.com/files/ftp_upload/1599/1599figure2.jpg">click here</a> to see a larger version of figure 2.
<img src="/files/ftp_upload/1599/1599figure3.jpg" border="0" /> Figure 3: qRT&#x2013;PCR analysis of neuronal marker gene expression in isolated da neurons (GAL421-7,UAS-mCD8-GFP) and the flow through fraction was performed in triplicate. The expression levels of the two neuronal-specific marker genes (elav and futsch) were assessed by qRT&#x2013;PCR. Values obtained from these analyses were normalized to the endogenous control (rp49), and the levels relative to those observed in flow through fraction were calculated using the &Delta;&Delta;C&tau; method6. Both elav and futsch were significantly enriched in the isolated da neuron population as compared to the flow through fraction.
<img src="/files/ftp_upload/1599/1599figure4tmb.jpg" border="0" /> Figure 4: Representative class-IV da neuron-specific Cy3 labeled microarray image file. Shown here is an Agilent Drosophila melanogaster whole-genome oligo microarray (4 x 44K) hybridized with Cy3-labeld total RNA isolated from class-IV da neurons purified by magnetic bead sorting. Please <a href="https://www.jove.com/files/ftp_upload/1599/1599figure4.jpg">click here</a> to see a larger version of figure 4.

<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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		<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>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>
		</div>
			<div>
			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 ...

isolation-purification-drosophila-peripheral-neurons-magnetic-bead

Research

JoVE Journal

Biology

13.3K 次观看.  George Mason University. 在这个视频文章中，我们目前的分离纯化的方法果蝇使用快速磁珠辅助细胞排序策略外周神经元。从分离的细胞获得的RNA可以很容易地用于下游应用，包括微阵列分析。

视频: Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...