1Department of Molecular and Microbiology, George Mason University, 2Krasnow Institute for Advanced Study, George Mason University
This article is a part ofJoVE General. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Iyer, E. P. R., Iyer, S. C., Sulkowski, M. J., Cox, D. N. Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting. J. Vis. Exp. (34), e1599, doi:10.3791/1599 (2009).
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°C until needed.
2. Selecting and Washing Larvae: (10-15 minutes)
3. Dissection: (10-12 minutes)
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.]
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.]
6. Magnetic Bead Cell Sorting: (45 - 75 minutes, dependent on antibody incubation time)
7. RNA Isolation from Magnetic Bead Sorted Cells: (60 – 75 minutes)
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 & 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.
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 μm 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 click here to see a larger version of figure 1.
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 click here to see a larger version of figure 2.
Figure 3: qRT–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–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 ΔΔCτ method6. Both elav and futsch were significantly enriched in the isolated da neuron population as compared to the flow through fraction.
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 click here to see a larger version of figure 4.
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 few parameters and using distinct GAL4,UAS-mCD8-GFP reporter transgenes which label the cell type or types of interest. Moreover, this protocol can be used in both loss-of-function and gain-of-function approaches where a gene of interest may be cloned into a UAS-mCD8-GFP transgene that can be coupled with a GAL4 transgene to direct either gene-specific loss-of-function (e.g. UAS-RNAi) or gain-of-function to a cell type of interest. For example, in the case of a transcription factor one may wish to identify potentially up- or down-regulated genes upon loss-of-function or gain-of-function expression in a cell type of interest. By isolating total RNA from the purified cell type of interest via this protocol and using this RNA to perform microarray expression profiling it is possible to identify differentially regulated genes that may represent downstream targets of transcriptional regulation that play a role in mediating phenotypic changes within the cell.
For successful cell sorting it is essential to give careful attention to the critical steps highlighted in the above protocol. Examples of common problem areas that may require some further troubleshooting and optimization, depending upon cell type, include (1) low cell yield and (2) cell clumping during magnetic bead isolation. In the first case, one may try reducing the concentration of Liberase Blendzyme 3, and compensate by increasing mechanical dissociation via douncing. In the second case, one may try reducing the magnetic field strength by applying a single or multiple layers of adhesive lab tape over the magnet.
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’s Office (E.P.R.I.).
|10X Phosphate Buffered Saline (PBS)||MP Biomedicals||PBS10X02||Diluted to 1X working solution|
|10X Liberase Blendzyme 3||Roche Group||11814176001||Diluted to 1X working solution (28 WĂĽnsch units/vial)|
|Biotinylated Rat anti-Mouse-CD8a antibody||Invitrogen||MCD0815||100 μg/ml stock concentration|
|BSA (Bovine Serum Albumin), Fraction V||GIBCO, by Life Technologies||11018-017||Prepare a 1% BSA solution in PBS|
|Dynabeads M-280 Streptavidin||Invitrogen||11205D||1 μl can bind 0.05-0.10 μg of biotinylated antibody|
|PicoPure RNA Isolation Kit||Molecular Devices||KIT0204||Follow manufacturer’s instructions|