This method can help to address key questions in neurobiology, such as motor neuron polarity, axon guidance, axon trafficking, and neurodegenerative mechanisms. The main advantage of this technique is to obtain a highly enriched motor neuron culture while we take to express our knockdown protein by minute affection. To begin this procedure, place one embryo in a dish of dissection media coded with silicon.
Under the microscope, hold the embryo with the abdomen against the silicon with forceps. With the second pair of forceps, insert one of the tips into the central canal of the rostrad spinal cord. Then, close the forceps to tear the dorsal tissue slightly.
Repeat this operation towards the connell side until the entire spinal cord is opened. Next, position the embryo so that the open spinal cord is against the silicon. Subsequently, detach the rostrad part of the spinal cord from the body.
Pinch the rostrad part of the spinal cord, and pull the embryo by the head to extract the spinal cord. Carefully pull away any remaining meninges and DRGs still attached to the spinal cord. Flatten the spinal cord on the silicon and using a scalpel, remove the dorsal half of the cord by cutting along the middle of each side.
Cut the middle part into ten pieces, and transfer them to a new tube. To prepare spinal cords cell suspension, collect the spinal cord pieces at the bottom of the tube. Replace HBSS with one milliliter of Ham F-10 medium and add ten microliters of trypsin.
Incubate the tube for ten minutes at 37 degrees Celsius. To stop the enzymatic digestion, transfer the fragments to a new 15 milliliter tube, containing 800 microliters of complete L-15 medium, 100 microliters of 4%BSA, and 100 microliters of DNAs and titrate, twice. Let the fragments settle for two minutes before collecting the supernatant in a fresh 15 milliliter tube.
Gently add two milliliters of 4%BSA at the bottom of the supernatant tube. Centro fuse it at 470 x gravity for 5 minutes. After 5 minutes, remove the supernatant and re-suspend the cell pallet with 2 milliliters of complete L-15 medium.
For motoneuron enrichment, split the cell suspension into two 15 milliliter tubes. Then, add two milliliters of L-15 PH medium to each tube. If starting with six embryos, use the equivalent of three embryos per tube, and three milliliters of medium.
Slowly add 2 milliliters of density gradience solution, to the bottom of each tube to obtain a sharp interface. Next, centro fuse it at 830 x gravity for 15 minutes at room temperature. After this step, small cells should be at the bottom of the tube, whereas large cells-such as the motoneuron-should be at the density gradient solution/medium interface.
Asperae 1 to 2 milliliters of the cells at the interface and transfer them to a fresh 15 milliliter tube. Adjust the volume to ten milliliters with L-15 PH medium. Subsequently, add two milliliters of the 4%BSA cushion to the bottom of the two tubes.
And centro fuse at 470 x gravity for 5 minutes at room temperature. After 5 minutes, remove the supernatant and re-suspend the pallet and 3 milliliters of complete L-15 medium. Repeat the centrifugate at 470 x gravity for 5 minutes at room temperature.
Then, remove the supernatant and re-suspend the cell pallet and two milliliters of complete culture medium. To culture motor neurons, dilute them to 5, 000-10, 000 cells in 500 microliters of culture medium per well in a 24 well plate. Then, remove the laminae solution with at P-1, 000 pipet tip.
Immediately transfer the motor neurons diluted in culture medium. to the coding plates to avoid drying. To prepare DNA, re-suspend 1 microgram of DNA in 50 microliters of neuronal culture medium.
And vortex for 5 seconds. To prepare the B tube, re-suspend 1.5 microliters of beads in 50 microliters of neuronal culture medium. Add 50 microliters of bead solution to 50 microliters of DNA solution and incubate for 20 minutes at room temperature.
During this incubation, withdraw 100 microliters of culture medium from the well that will be transfected. Subsequently, transfer 100 microliters of the DNA bead mix to each well. And incubate the 24 well plate, 20-30 minutes on the magnetic plate at 37 degrees Celsius.
Shown here, is the modem neuron morphology revealed by standing of endogenous non-phosphorylated neurofilament heavy chain after four days of culture. In these images, show the modem neuron morphology after four days in vitro and transfected for EGFP trans gene by magnetofection at day two. Motto neurons were counter-stained with the marker SMI-32 or the pan-neuro marker TUJ-1 and the arrows indicate the soma of the neurons.
These are their confocal images of magneto fected motor neurons, expressing wild type or mutant EGFP NEFH constructs and stained for their autophagic marker LC-3B. Arrows show the mutant EGFP NEFH protein aggregates that are also LC-3B positive Following this procedure, montoholda curcha can be analyzed by video microscopy to visualize axon trafficking and add some guidance. After its initial development to address fundamental questions in neural biology, this technique also prove to be a powerful tool to explore physio pathological mechanisms implicating the spectrum of motoneuron disorders, such as toxic shock syndrome disease, samuel trophic lateral sclerosis, or spinal muscular atrophy.
