This video demonstrates a method to dissect and culture comm choral neurons from embryonic spinal cord. The procedure begins by harvesting E 13 rat embryos and then dissecting out the spinal cords. During the dissection, spinal cords are opened along the roof plate and prepared in the open book configuration.
Dorsal tissue strips are dissected and collected in a tube. The dorsal tissue is dissociated into a single cell suspension by trypsin and mechanical disruption. Commissural neurons are then cultured on polylysine coated glass cover slips or tissue culture dishes.
They can then be used to study the cellular and molecular mechanisms of axon growth and guidance. Hi, I'm Sebastian from the Laboratory of Molecular Biology of neural Development at the Clinical Research Institute of Montreal. Hi, I'm Steve Moen, also from the Molecular Biology of Neural Development Laboratory.
Today we will show you a procedure for dissecting and culturing commissural neurons from embryonic spinal cord. We use this procedure in our laboratory to study the cellular and molecular mechanisms of accent guidance. So let's get started.
Today, spinal cords will be dissected from freshly harvested E 13 rat embryos placed in a dish of L 15 medium throughout the dissection. It is important to keep embryos and solutions on ice. Remember that for all dissection steps, it is crucial not to nickel stretch the spinal cord as this can impede the isolation of a fully intact spinal cord.
Under a dissecting microscope, separate the embryo from all extra embryonic tissues and embryonic membranes. Then place one embryo in the dissection dish. Use micro scissors to cut away the head and posterior parts of the angles shown here.
This will create a flat surface that will help position the embryo for easy access to the spinal cord. Position the embryo ventral side down with the anterior pointing away. Use forceps to firmly pin down the embryo on one side.
Do not pinch or release the forceps or the tissue will tear with the second pair of forceps. Grasp the skin to the side of the spinal cord. Twe the tips of the holding forceps.
Peel away the skin covering the back of the embryo to expose the spinal cord wrapped in its meninges to partially detach the tissue to the right of the spinal cord. Use closed forceps to poke the tissue starting at the level of the holding forceps. Get as close as possible to the spinal cord without touching it.
Repeat several times along the spinal cord. This would attach some dorsal root ganglia and disrupt ventral organs. Leave some tissue attached at the anterior and posterior ends starting from the anterior end.
Use a hook shaped tungsten needle to cut the meninges and open the spinal cord along the roof plate. Rotate the embryo 180 degrees and disrupt the tissue from the remaining side. Using the same method.
Then completely detach the tissue on the left side of the spinal cord, the side that was disrupted earlier originally on the right place the spinal cord on its side and use forceps to remove most of the remaining mesenchymal tissue and dorsal root ganglia. Use forceps to peel open a short flap of meninges. At this point, the meninges and spinal cord are two sheets of sandwich tissue.
Use the forceps to pin down the larger portion of the spinal cord at the anterior end. Grasp the two separated segments with forceps and peel off the meninges. With a smooth constant movement, uneven peeling can break the meninges layer and or the spinal cord.
If the meninges layer breaks during pulling, the portion of the spinal cord that remains attached to the meninges is usually unrecoverable. Although it is sometimes possible. Use a plastic pipee to gently transfer the isolated spinal cord to a dish of L 15 medium with 10%heat inactivated horse serum on ice before processing more embryos.
Remember to change the medium in the dissection dish with chilled L 15 to keep the embryo cool and help preserve tissue integrity. Harvest spinal cords from all the embryos before proceeding to the dorsal spinal cord dissections. To begin the dorsal cord dissection, place one freshly isolated spinal cord in a Petri dish containing L 15 with 10%heat and activated horse serum.
Cut away the wider anterior portion, which includes part of the hind brain. Then lay the spinal cord flat and spread out like an open book. The dorsal tissue is located in the most lateral parts of the open book spinal cord while pinning the cord with a straight tungsten needle using L-shaped tungsten needle to cut out a strip approximately one fifth, the width of one half of the spinal cord cutting wider strips will increase yield, but reduce the purity of the culture.
Transfer multiple dorsal strips to a 15 milliliter conical tube containing L 15 medium with 10%horse serum and place the tube on ice. The rest of the procedure will take place near or inside the tissue culture hood. For these cultures, it is important to prepare solutions and equipment in advance, usually before dissection.
The tation step uses fire polished pasta pipes of two sizes, one of a diameter, which is half the original size and the other slightly smaller. Before starting coat the two fire polished pipes with serum containing medium by aspirating medium into the pipes and letting sit for 30 seconds. This will reduce the number of cells that stick to the glass during ation.
Begin by verifying that the dorsal strips have settled to the bottom of the tube. Under lamina flow hood, remove most of the SUP natant with a plain pasta per pet. Quickly wash with three milliliters of cold magnesium and calcium free HBSS.
Let the dorsal neural tube strip settle for two minutes. Then remove the HBSS with the pasta. Bring the volume up to 4.7 milliliters.
With warm HBSS, add 0.3 milliliters of 2.5%trypsin to give a final concentration of 0.15%Tryin and mix gently incubate in a 37 degree Celsius water bath for seven minutes. Mixing once gently halfway through the incubation. Once the incubation is up, add DNAs for a final concentration of 150 units per milliliter at magnesium sulfate for a final concentration of 0.15%and mix briefly.
The dorsal neural tube sections should now be fragmented. Centrifuge the tissue fragments at 200 G for four minutes. Remove the SUP supernatant with a pasta perpe leaving about 50 to 100 microliters of liquid at the bottom of the tube.
Flick the tube gently to loosen the pellet. Then wash the cells by adding five milliliters of warm HBSS. Let the cells settle at room temperature for two minutes.
Spin down and wash the cells again. This time, resus suspending in two milliliters of warm HBSS. Use a half diameter fire polish pipet to triturate the cells by slowly pipetting up and down four to six times, pipee the liquid against the side of the tube to break up larger clumps to reduce cell death.
Avoid making bubbles and do not overate. Use the smallest fire polish glass pasta perpe to further dissociate the cells by slowly pipetting up and down three to four times in the same manner. It is not necessary to dissociate all the cell clumps as aggregates tend to form of dead cells in debris.
Let any tissue fragments settle for one minute. Then remove 20 microliters of the SUP natant, which should contain suspended cells for counting in a hemo soter, the neurons should be at least 95%viable by trian blue ion plate. The neurons in neuro basal plating media at a concentration that is appropriate for your application.
16 to 18 hours later, change the medium to neural basal growth media to avoid dislodging. The neurons gently remove the media with a pipet rather than aspirating with a vacuum pump. Here are some representative results of isolated commissural neurons plated on PLL Coated glass cover slips four hours after plating neurons have adhered to the surface 30 hours following plating.
Most neurons have extended axons with a visible growth cone. These neurons can be easily used in immunofluorescence experiments. In this example, microtubules are labeled in green f acton in red and NA in blue.
We've just shown you how to dissect and culture commissural neurons from embryonic spinal cords. When doing this procedure, it's important to remember that the purity of the commissural neurons depends on the thickness of the dorsal strip that are dissected out. So that's it.
Thanks for watching and good luck with your experiments.
