This procedure begins with isolating the superior cervical sympathetic ganglia from P zero neonatal rats. Each brain has two superior cervical sympathetic ganglia. Approximately 24 ganglia can be harvested from a single litter of rat pups.
The dissociation of the ganglia begins with trips and treatment for 30 minutes. Further dissociation is accomplished via repeated reation with a fire polished pester pipette resulting in a single cell suspension. Sympathetic neurons are then plated on collagen ENC coated dishes.
Hi, I am Nila Zurin from the laboratory of Dr.Lloyd Green in the Department of Pathology and Cell Biology at Columbia University City. Today we will demonstrate how to isolate and culture primary sympathetic neurons from superior cervical ganglia of newborn red pups. In our lab, we typically use these cells to study apoptosis in response to nerve growth factor withdrawal.
So let's get started. We begin the procedure by coating a culture dish with rat tail collagen 24 hours before performing the dissection. Depending on the nature of the experiment, a 24 well or 48 well culture plate is used.
Next solutions media and equipment will be prepared. These materials and reagents can be found in the text protocol that accompanies this video. It's important to note that the uridine and five FDU solution be made just before use.
Continue preparation by fire. Polishing the glass pasture pipettes for the tation steps. The tip of the glass pipette is held in the flame for a few seconds while rotating to narrow and smooth.
The opening fire polishing should be conducted in a sterile cell culture hood. To begin the dissection, collect P zero to P one neonatal rat pups. A neonatal rat pup is quickly wiped with 70%ethanol to reduce contamination.
Following decapitation, the head is placed on the dissection pad with the ventral side facing up and the coddle end oriented toward you. The severed trachea should be readily visible. Sterile syringe needles are used to secure the severed head to the dissecting pad in the following manner.
Push one pin through the roof of the mouth into the pad and push the second pin through the severed spinal cord into the pad. After moving the dissecting pad under the scope and looking through the oculars, use two pairs of forceps to clear away the skin and fat around the trachea. Taking care not to go too deep, a pair of muscles running almost parallel to each other will be exposed on either side of the trachea.
During the dissection, you'll need to irrigate with cold sterile PBS or cold serum free RPMI 1640 medium delivered with a sterile pasture pipette to wash away excess blood. This may be done once or more than once, depending on how fast one works. The forceps are then used to cut and remove the muscle on one side first.
Once the muscle is removed, the carotid artery will become visible. This artery runs alongside the trachea and bifurcates into what looks like a Y shaped ribbon. This bifurcation is an important landmark and once it is located, it will be relatively easy to find the superior cervical ganglia.
The artery is severed at the most coddle end and lifted up slightly with the forceps. The other end of the artery is kept attached. Once this is done, the superior cervical ganglion will become visible.
One should see that it is an almond shaped, glossy looking small mass of tissue loosely attached to the artery at the point of bifurcation, and that it has thread like pre and or post ganglionic connectives. A second pair of forceps is used to gently detach the superior cervical ganglion and place it in a small 35 millimeter culture dish containing cold serum free RPMI 1640 medium. Next, the ganglion is extracted on the other side of the trachea following the same protocol.
Once all the ganglia have been dissected out, they're freed as much as possible of extraneous tissue. Looking through the scope, you can notice that the ganglia have attached pieces of carotid artery fat or other tissue forceps are used to tease away these unwanted materials. The pre and or post ganglionic connectives are also trimmed away.
Each ganglion has about 10, 000 neurons. Once the neurons are plated, they will regrow their neurons cleaned. Ganglia are then placed into a sterile 15 milliliter conical polypropylene tube containing RPMI 1640 media.
Next, the sympathetic neuron culture is established. The ganglia are centrifuge at 200 G for two minutes and the snat is discarded. The ganglia are resuspended in 0.5 to one milliliter of 0.25%tripsin solution, and then placed in a 37 degrees Celsius water bath or incubator for 30 minutes to help dissociate the cells in the ganglia.
After 30 minutes, 10 milliliters of complete medium are added in order to dilute out and neutralize the trypsin. The solutions are then centrifuge at 200 G for two minutes. The super agent is then discarded and the ganglia are resuspended in two milliliters of final medium.
Using one of the pre-prepared fire polished pipette tips with a wide opening, the cells are further dissociated by tri the ganglia up and down 20 times. The tube is then placed on ice for a few minutes. Next, the pipette is fire polished again to make the opening even narrower.
After the pipette cools, the ganglia are triturated 20 times more. The medium will become progressively more cloudy, indicating that the cells are being dissociated. This step is repeated once or twice more depending on how well the cells dissociate.
After some time, the ganglia will have dissociated into neurons and no intact ganglia will be visible in the tube. There may be some tissue material that will not dissociate at this point. The tube is left to sit on ice for a few minutes so that any unassociated debris can settle to the bottom.
All but about 50 microliters of the supernatant is carefully collected and placed in another tube. 50 microliters is left at the bottom of the tube to prevent transfer of unassociated debris. More of the final medium is added to the tube containing dissociated cells.
The volume of final medium added depends on the type of culture plate. One is using to culture the neurons. For example, if a 24 well dish is to be employed, then plate one ganglia per well in 0.5 milliliters of final medium per well.
A typical rat litter consists of 12 pups, which would give 24 ganglia. This is enough to seed 1 24 well plate, and the total volume of final medium is 12 milliliters. The use of uridine five FDU in the final medium will prevent the proliferation and promote the death of a small population of glia and other cell types.
Present in the culture, cells are fed every 48 hours by replacing two thirds of the medium. With fresh RPMI plus 1%horse serum plus NGF and uridine and five FDU. At first, the culture contains sympathetic neurons that look round with no neurons.
Short neurons become visible 24 hours after dissection and progressively become denser and more branched over time. The we've just shown you how to isolate and culture primary sympathetic neurons from superior cervical ganglia of newborn rat tubs. When doing this procedure, it's important to remember to one, take time and care cleaning the ganglia, remove all debris, blood vessels, and fat.
This step is important in ensuring a culture that is more or less homogeneous and free of extraneous cell types. Two, during the dissociation step, be patient and take the time to separate the neurons so that they're not found in large clumps. Once they're seated onto the plates, large clumps will make it difficult.
In downstream applications such as transecting or infecting the neurons, immuno staining may also be hindered, and any experiments that require counting the neurons will be difficult to carry out as well. Lastly, it is not necessary to supplement the medium with penicillin streptomycin. Every time the cultures are fed additional penicillin streptomycin, the very first time the cells are plated is sufficient.
Do add fresh uridine five FDU to the medium every time the medium is exchanged. Our lab has used sympathetic neuronal culture to study apoptosis in response to nerve growth factor withdrawal. So that's it.
Thanks for watching and good luck with your experiments.
