This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.
1. Preparation for the dissection:
2. Dissection of Superior Cervical Ganglia:
3. Cleaning the Ganglia:
4. Establishing Sympathetic Neuron culture:
We use Sprague Dawley rats for our cultures.
Take time and care when 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. The addition of uridine/5-FDU will largely eliminate any remaining non-neuronal (mitotic) cells contaminating the culture or at least suppress their proliferation.
In addition, during the dissociation step, be patient and take the time to separate the neurons so that they are not found in large clumps once they are seeded onto the plates. Having large clumps of cells will not yield good experimental results. For example, large clumps will make it difficult for the neurons to be transfected or infected. Immunostaining may also be hindered and any experiments that require counting the neurons will be difficult to carry out, as well.
It is not necessary to supplement the medium with pen/strep every time the cultures are fed. Addition of pen/strep the very first time the cells are plated is sufficient. Do add fresh uridine/5-FDU to the medium every time the medium is exchanged.
We have used sympathetic neuronal culture to study apoptosis in response to NGF-withdrawal. For example, in Biswas et al. 2007 (2), sympathetic neurons were transfected with shRNA against several molecules that play important roles in promoting apoptosis. Deckwerth et al. 1996 (5) have used sympathetic neurons from wildtype mice and mice lacking the pro-apoptotic gene, Bax. They have looked at how these neurons behave when they are deprived of NGF. Figure 6 from Biswas et al. 2007 (2) and Figure 3 from Deckwerth et al. 1996 (5) show what transfected and non-transfected sympathetic neuronal cultures look like, respectively.
The authors have nothing to disclose.
NZ would like to thank lab members Subhas Biswas, Andrew Sproul, and Ryan Willet for training her in dissecting and harvesting sympathetic neurons. Supported by grants from the NIH-NINDS.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Forceps, Stainless steel #5 | Tool | Roboz | RS4976 | |
Scissors, DEAVER straight sharp-blunt- 43mm blades – 5.5 | Tool | Roboz | RS6760 | |
RPMI 1640 w/ L-Glutamin | Reagent | Cellgro, Mediatech, Inc. | 10-040-CV | |
Fetal Bovine Serum | Reagent | SAFC Biosciences | 12103c | |
Donor Horse Serum | Reagent | JRH Biosciences | 12449 | Heat-inactivate by incubating in 56°C waterbath for 30 minutes. |
Penicillin/streptomycin | Reagent | Gibco, Invitrogen | 15140-122 | |
Trypsin without EDTA | Reagent | Difco | MT 25-050-CI | |
Uridine | Reagent | Sigma | U3003 | |
5-Fluoro-5′ deoxyuridine | Reagent | Sigma | F-8791 | |
NGF | Reagent | Harlan Bioproduct | BT-5025 | |
Dissection Microscope and light source | Microscope | |||
15 ml polypropylene tubes | Tool | BD Falcon | 352096 | |
Cell culture dishes | Tool | Corning, Nunclone |