This procedure begins with making a scratch with a pin rake onto the center of a collagen coated plate, and pipetting 30 microliters of Methylcellulose onto the center of the scratches. Grease each divider using a grease loader and attach it to a collagen coated plate plate. 100, 000 cells from E 15 rat embryos into the middle compartment.
After nine days, the cells will have extended axons into the side compartment and will be ready for experimentation. Hi, I'm Maria Murphy from the laboratory of Dr.Rosalyn Siegel in the Department of Pediatric Oncology at the Dana-Farber Cancer Institute. Today we will show you a procedure for the preparation and maintenance of Dosa root ganglia neurons in compartmented cultures.
We use this procedure on our laboratory to study neurotrophin signaling. So let's get started. Before setting up the compartmented chambers, P 35 tissue culture plates must be coated with collagen.
To do this, add one milliliter of a 0.71 milligram per milliliter solution of collagen in 0.001 normal HCL to the plates and incubate them at 37 degrees Celsius for two days. After two days, make a scratch in the middle of the collagen coated plates with a pin rake. Use an outward motion.
Then place 30 microliters of the N two Methylcellulose on the middle of the scratch. Set the dishes aside. Next, attach a 23 gauge lure stub adapter to the grease loader.
Grip the Teflon divider with a pair of 90 degree angle hemostats and lay it flat with the divider facing up under a microscope. Looking through the scope, race the divider with grease. Make sure that each time the adapter is placed at a new starting point, it is inserted into the grease from the previous step so that there is a continuous line of grease.
Once the grease is applied to the entire divider, turn one of the prepared P 35 dishes upside down and place it so that the N two Methylcellulose is over the middle compartment. Press down on the bottom of the dish with a pair of tweezers. Make sure to press on the inside of the divider in four corners.
It is important to press firmly enough so that the grease makes a complete seal with a dish. But if too much pressure is added, the axons will not cross into the side compartments. Next, pick up Hemostats.
Turn over the dish and unclamp the divider. Then place the dish with the divider firmly attached under the microscope, focusing on the bottom of the middle compartment. Use the grease loader to make a small barrier connecting both sides of the divider about a quarter of a centimeter high.
This step will prevent the cells from leaking out once they're placed in the middle compartment. After setting up several cultures, place DRG media in each of the side compartments. Then place in the incubator where the cells will be maintained.
Allow the cultures to sit for several hours and then check for leakage. If the media has leaked into the middle compartment, then the culture is unusable. When first learning this technique, it is important to set up more cultures than are needed for an experiment, as several will probably be leaky.
After confirming that the compartmented chambers are usable, proceed with the maintenance of the dorsal root ganglia neuron culture. Prior to experimentation, the DRG neurons are maintained in the compartmented cultures. Over a period of nine days at 37 degrees Celsius on day one, replace the DRG media in the side compartments with 100 nanograms per milliliter.
DRGN media plus A-C-D-R-G-N media contains both nerve growth factor and brain derived neurotrophic factor. The recipe for this media is available in our written protocol. Perform the dissection on E 15 rat embryos and add 100, 000 cells to the center compartment.
On day two. Add 10 nanograms per milliliter DRGN media plus a C to the outside of the Teflon divider until the media flows over the grease barrier and exchanges fluid with the center compartment on day three, replace media in the side compartments with 100 nanogram per milliliter. DRGN omitting the a c.
Also replace the surround with 10 nanogram per milliliter. DRGN omitting the RSC on day six. Replace media in the side compartments to one nanogram per milliliter, DRGN plus RSC and the surround with DRG media plus RSC.
On day nine, the compartmented culture is ready for experimentation. After experimentation is completed, the Teflon dividers can be reused, but must be properly cleaned. To do this, remove the divider from the plate and wipe off all of the remaining grease.
Then place in sulfuric acid for two days. After two days, remove dividers from the acid and rinse them with water three times, then boil for 20 minutes and allow to dry. Place the dry dividers in a glass P 100 Petri dish and autoclave for 20 minutes after sterilization, they'll be ready for future experiments.
This compartmented culture system allows separation of the cell body from the axon in order to study mechanisms by which neurotrophins signal across long axons. Since there is fluidic isolation between the compartments, it allows for selective stimulation or treatment of one compartment without the other compartments being affected. We've just shown you how to prepare and maintain dosa root ganglia neurons in compartment to cultures.
When doing this procedure, it's important to remember to press gently when sealing the grease divider to the collagen coated plate. Aspirate the liquid from the top of each side compartment when changing the media as to not disturb the axons. Never change the media directly from the middle compartment when feeding the neurons.
Change only the media from the surround and let it flow over the grease barrier into the middle compartment. So that's it. Thanks for watching and good luck with the experiments.
