Drosophila larvae bearing a neuron specific CD eight GFP Fusion Reporter transgene are used to isolate DA neurons. The larvae are dissected by inverting their cuticle inside out, exposing the peripheral nervous system. The inverted larvae are subjected to enzymatic dissociation, followed by mechanical dissociation via vortexing, ation and downing.
Cells are then filtered to obtain a single cell suspension containing primarily epithelial muscle and neurons. Antibody coated magnetic beads are added to the cell suspension where they bind neurons expressing the CD eight tagged GFP fusion protein. Finally, the magnetic bead coated neurons are isolated by placing the solution in a strong magnetic field yielding highly purified neuron populations.
Hi, I'm Dan Cox from the Department of Molecular and Microbiology in the Krasno Institute for Advanced Study at George Mason University. Hi, I'm Ish AER from the laboratory of Dr.Dan Cox. Today we will show you a procedure for the isolation and purification of DLO peripheral neurons by magnetic bead sorting.
We use this procedure in our laboratory to study the molecular mechanisms regulating class specific and right morphogenesis. So let's get started. To begin this procedure, pick 30 to 50 age matched third in star larvae and placed them in a 1.6 milliliter micro fuge tube containing one to 1.2 milliliters of one XPBS.
Close the tube and vortex it at the maximum setting three times for one second each. Using a fire polished past your pipette, discard the supernatant completely. Add one milliliter of PBS and repeat the wash and vortex steps three times until the supernatant is visibly clear of any food particles and debris.
Briefly repeat the wash with one milliliter of 70%ethanol. Then wash the larvae twice with one milliliter of double distilled water. And finally, repeat the wash once again with one milliliter of R nase away and three times in one milliliter of double distilled water for complete removal of the RNA away the larvae are now ready for dissection for dissection.
Place 10 to 12 larvae on the center of a sill guard coated 35 millimeter Petri plate. Position them slightly away from each other and discard any larvae that do not seem to be at the appropriate developmental stage. Next, use a pair of fine dissection scissors to cut open the anterior tip of the larvae using a pair of dull dumont.
Number five, forceps. Invert the larvae inside out. Start by inserting 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. Pressing the cuticle down on the syl guard surface makes it easier. Using the second pair of forceps, push the larval cuticle inside out.
Make sure the larvae are completely inverted so that all the soft tissues are exposed to the solution For easy dissociation. Practice this a few times before attempting to dissociate the cells. After dissecting three to four larvae, transfer them immediately to fresh ice cold PBS in a micro fuge tube.
Repeat until the required 30 to 40 larvae are collected before proceeding to remove non-specific cells. Keep in mind that 10 to 20%loss during dissection and association is expected now that all the larvae are dissected. Clear loosely adherent non-specific tissues such as fat bodies and central nervous system cells.
By replacing the snat of the tube containing the inverted larval cuticles with 700 to 800 microliters of fresh ice cold PBS. Lastly, pulse. Vortex the micro fuge tube five times for three seconds per pulse at full speed.
Then discard the supernatant and replace it with approximately 700 to 800 microliters of fresh ice. Cold PBS. Repeat this step three times and finally resuspend the larval cuticles in 400 microliters of fresh ice.
Cold PBS before moving on to dissociate the cells to isolate the dendritic arborization or da larval neurons. Use a combination of enzymatic and mechanical dissociation. Beware of over dissociation which may cause the loss of the cell surface marker leading to poor cell yield and low cell viability.
Start the dissociation by adding 1.5 microliters of one x liase zyme three at 28. Once units per vial to the larval cuticles suspended in 400 microliters of PBS. Vortex the solution two to three times for one second each at maximum setting to remove the loosely adherent cells from the cuticles into solution.
Then incubate the solution at room temperature for five minutes. Since incubation time greatly affects the final cell sorting efficiency. Do not exceed 15 minutes at the end of the incubation pulse.
Vortex the tube 20 to 30 times at full speed for two seconds per pulse. This should release the muscle and other non-specific tissue into solution. Inspect the sample periodically under a fluorescence enabled stereo microscope to determine if the peripheral neurons are still attached to the cuticle.
Inspect a small sample from the solution by observing the micro fuge tube directly under a fluorescence enabled stereo microscope to determine the level of dissociation. Continue to do this at each step of the dissociation stop vortexing before the peripheral neurons appear to dissociate from the cuticle into the solution. Briefly wash the larval cuticles with two to three exchanges of one milliliter each of fresh ice cold PBS.
And finally, resus. Suspend them in 500 microliters of fresh ice cold PBS containing 1%BSA To continue with the mechanical dissociation of the cells preco A two milliliter conti tissue grinder and large clearance pestle with a 1%BSA in PBS solution. And after a brief rinse, discard the BSA solution.
This will prevent the larval cuticles from sticking to the glass surfaces. Subsequently pre-cool the tissue grinder and pestle by placing them on ice for a few minutes In order to prevent cell damage or lysis. Then use a fire polished past your pipette to transfer the cuticles to the tissue grinder.
Now down the tissue with slow and steady strokes avoiding frothing for approximately 20 to 30 strokes. Make sure to down slowly and steadily. Otherwise the cells may lice.
To assess the cell dissociation levels, wipe the wall of the tissue grinder with a clean Kim wipe tissue and inspect under a fluorescent stereo microscope. The neurons should have detached from the cuticle and can be seen in the solution. 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 in completely dissociated cells downs further until the cells achieve a single cell suspension. Continue the mechanical dissociation using a fire polished past your pipette. Narrow to approximately 50%of the standard tip diameter to rate the solution five times followed by 10 times with a fire polished past your pipette.
Narrow to approximately 25%of the standard tip diameter. Note that forceful tation may damage the cells. Monitor the cells between steps and adjust the procedure accordingly.
Finish by filtering the solution through a 30 micrometer cell filter and collect the cell filtrate in a 1.6 milliliter micro fuge tube. The resulting solution should consist of a single cell suspension and is now ready for magnetic cell sorting. Magnetic beads coated with an anti CD eight antibody.
Were prepared as described in the accompanying written protocol and the DA neurons express a CD eight GFP fusion protein targeted by the antibody. Start the cell sorting by adding 15 microliters of antibody coated magnetic beads to 500 microliters of cell suspension. Incubate the cells with antibody coated magnetic beads for 30 to 60 minutes on ice with occasional hand mixing incubation at a higher temperature or longer time may result in non-specific antibody binding.
Next place the micro huge tube in a strong magnetic field for two minutes. All positively selected cells along with unbound beads will adhere to the side of the tube, slowly aspirate the supernatant, disturbing the cell pellet, wash the cells three to four times in fresh ice cold PBS. This step removes any remaining nonspecific cells.
Finally resuspend the cells in 30 microliters of fresh ice cold PBS to estimate the purity and yield of cells. Pipette five microliters of the cell suspension onto the polished surface of a hemo cytometer 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. Finally, use the pico pure RNA isolation kit to isolate RNA from the cells bound to the beads. Add the extraction buffer to the sorted cells, but remove the beads using a magnet prior to the column purification step, use one microliter of the extracted RNA to assess total RNA quality on an Agilent Bioanalyzer 2100.
The RNA purified from the isolated DA neurons is of excellent quality as indicated by the presence of sharp 5.8 s ness and 28 s ribosomal RNA peaks when analyzed on an Agilent 2100 Bioanalyzer, beginning with 30 to 43rd in star larvae, we were capable of isolating on average 300 to 500 class four DA neurons using the PPK gal four driver and 1500 to 2000 DA neurons classes, 1, 2, 3, and four. Using a panda neuron specific gal four driver quantitative R-T-P-C-R with L 11 fch, which are neuronal specific markers revealed significant fold enrichment of both indicating a highly specific enrichment for DA neurons using the protocol we demonstrated as compared to flow through. Finally, the isolated RNA from both panda neurons and class four DA neurons was used to perform transcriptional expression profiling on Agilent Drosophila Melanogaster whole genome oligo microarrays, identifying many factors implicated in da neuron morphogenesis.
We has shown you how to isolate and purify drosophila peripheral neurons via magnetic bead sorting from the third instar stage of larval development. When doing this procedure, it is important to balance the enzymatic and mechanical dissociation steps. Excess enzymatic dissociation may lead to the degradation of CD eight cell surface face markers leading to inefficient or failed Cell sorting.
It is also important to maintain RNA free conditions throughout the procedure in order to preserve the integrity of the purified RNA from the isolated neurons. So that's it. Thanks for watching and good luck with your experiments.
