Hi, I'm Lauren Frank from the Laboratory of Emmanuel Pothos in the Pharmacology and Experimental Therapeutics department at Tufts University's School of Medicine. And I'm Emmanuel Pothos. And today we will show you a procedure for generating primary midbrain dopamine cell cultures from rodent neonates.
This procedure includes the following steps, preparing tools and reagents. Dissecting the midbrain of P zero to P two rodent brains tating the brain segments and plating the dissociated neurons. So let's get started.
This procedure, there are a number of preparation steps that must be performed in advance two to three weeks before generating the dopamine neuron culture, glial cells must be plated so they have time to proliferate and cover the bottom of the dishes. The primary dopamine neurons will be cultured on a monolayer of glial cells one to seven days before the primary neuronal cell culturing. Remove the media from the glial cells and replace with two milliliters of fresh neuronal medium the day before AC culturing all items except the dissection instruments must be sterilized using UV light including 12 dissection plates.
Two dissociation vials with a micro magnetic stir bar. Two vial caps with two small holes poked in the top. Slide rings spread out.
And the slide ring box, remember to leave it opened. All of these tools are coated with 70%ethanol before beginning on the day of culturing. First, take all of the constituents for the pepane solution that will be used to dissociate the primary neuron solution from the freezer and allow them to thaw.
Set up a hot plate to heat a beaker of water to 34 degrees Celsius for the digestion and prepare carbogen saturated water for oxygenation of the pepane solution. During the digestion, 15 sterile tubes filled with four to six milliliters of sterile PBS for washing of the brains prior to dissection are prepared and put on ice. 20 to 30 milliliters of fresh neuronal medium are allotted and placed in the incubator.
Next, the dissociation vial is set up by using a sterile flip filtration unit to filter the pepane solution, which is then transferred to the dissociation vial with a 25 milliliter strep cap the vial, insert a sterile syringe, throw a hole in the cap, which is connected to a 0.2 micron filter. Connect the carbogen into the filter and para film this in place. Place the vial in the styrofoam disc.
Once the dissociation vial is in place, make sure the magnetic stir bar is moving. The gas is making it into the vial, and the temperature is stabilized at 34 degrees Celsius. The dissection scope is brought into the hood and the dissection instruments are laid out on aluminum foil.
Then lay out one large square of aluminum foil and 12 smaller squares. Leave a pair of scissors for decapitations with the aluminum foil. Place a styrofoam box with ice water outside the hood.
Next, prepare the anesthetic, which is a mixture of 0.075 milliliter ketamine and 0.075 milliliter xylazine. Then obtain at least 12 P zero to P two pups for 135 millimeter plates with a 23 millimeter glass. Well for mice, make sure there is a genotype match for the whole litter.
If the genotype is unknown, cells from each animal will need to be plated on separate dishes and accurate records will need to be kept to match the cells with subsequent genotyping results. To begin the dissection, anesthetize the first animal with an intraperitoneal injection of ketamine Xylazine. When animal does not respond to a tail flick test, put it in ice water for 30 seconds until hypothermic.
Then rinse one small aluminum foil, square the decapitation scissors and animal head with 70%ethanol to avoid contamination of the brain tissue during the dissection, decapitate with the scissors, allow the head to fall onto the aluminum foil square and move it under the hood. Gently remove the brain by first cutting the skin with the small scissors to expose the skull. Cut around the skull 360 degrees.
Then peel away the skull cap with forceps. Lift the brain out of the skull using a small spatula and place it into a 15 milliliter tube of ice. Cold PBS put the tube back on ice before removing the next S brain.
Repeat the dissections until there are three brains in separate tubes on ice. Pour one brain and PBS onto the first dissection dish. Under the microscope, place the brain onto a platform of sill guard glue as a cushion so the blade stays sharp.
Use a transfer pipette to put the segments into a fresh tube of PBS to wash the segments before transferring to the pepane solution. Start a timer When the first segments go in, continue the dissections until all brains are being digested. In pap pain.
The first brain segments will have been digesting for about one hour. By the time the last segments are added, allow the digestion to proceed for another hour during the digestion. Make sure the temperature in the pepane bath is stabilized at 34 degrees Celsius.
To begin the tation step, use a transfer pipette to place the brain segments in a sterile 15 milliliter tube. Try to avoid bringing over excess pepane solution. However, if this occurs, then remove excess pepane.
Next, wash the segments by adding two milliliters of warmed neuronal medium from the incubator. Allow segments to settle and then carefully discard as much solution as possible without disturbing the segments. Perform a total of three washes.
Remember to change pipettes between washes to avoid contamination using a fresh transfer of pipette. Begin iterations in two milliliters of neuronal media. Triturate 25 times, avoid letting in air bubbles.
Then let the unassociated segments settle for three minutes. Use a transfer pipette to collect as much super natin as possible without disturbing segments at the bottom. Keep the super natin in a new sterile 15 milliliter tube.
Repeat the previous step using a 1000 microliter pipette tip and then a 200 microliter tip, after which the segments should appear completely dissociated to the eye. The supernatant from all alterations is collected in the same 15 milliliter tube. We are ready to plate The cells finally iterate 10 times with a transfer pipette to achieve homogeneous cell suspension for counting.
Using a sterile yellow pipette tip covering each tip of the forceps. Carefully drop a slide ring as centered to the glass as well as possible. Inside each dish, these rings will be used to isolate an area within the culture dish where the neurons will be plated.
Use the pipette tip to slide rings over the center well of each dish. And gently add the appropriate volume of cells to the dish. Add the cells to the center of the ring that you have already placed in the dish.
Remember to switch tips for every dish. Next, add a hundred microliters of diluted GDNF solution as close to the outside of the ring as possible of each dish to aid in the proliferation of neurons. Next, the mitosis inhibitor fluoro dioxine or FDU is added outside the ring to prevent glial cells from overgrowing change.
Pipette tips often return to the incubator and allow cells to settle overnight the next day using sterile pipette tips to cover the tips of the forceps, remove the rings, return the dishes to the incubator and disturb them as little as possible until it's time for experiments. Check dishes daily for possible contamination, and if it is observed, remove the problematic dishes immediately. Cultures are ready for use in electrophysiology, molecular biology experiments and immuno cyto within three weeks.
Now that the cells have been plated, let's look at some representative examples of the cells in culture. Here we're looking at a primary culture that was plated 14 days ago. This is an example of a good culture because you can see distinct cell bodies with processes leading off of them.
In addition, note the absence of any mold or fungal growth in the dish that would indicate an infection. We've just shown you how to generate primary dissociated mid-brain dopamine cell cultures from rodent neonates. When doing this procedure, it's important to remember, maintain a sterile environment, use only neonates P zero to P two plate on healthy glial substrates with glial conditioned media, maintain a consistent plating density.
So that's it. Thanks for watching and good luck with your experiments.
