Hi everybody,
I want to establish a protocoll for isolating and culturing primary rat neurons from E16-18 rats. The culture does not have to be neurons-only, but should be reproducible in terms of composition of cell types. The simpler the protocoll is, the better.
The cells are later going to be used to establish patch-clamp experiments.
I am specific about E16-18 prenatal rats because we are isolating DRG-neurons from those animals and I would like to use the same animals for my brain-cell-isolation.
First I had the idea to isolate cerebellar granule neurons (found an easy protocoll for that), but it seems these are not yet developed until birth?
I am looking forward to your comments and ideas, thanks in advance!
Cheers,
Jörg
Hi Jörg,
Of cause it all depends of what you want to study, but in your case you may want to consider hippocampal or cortical neurons. These cultures are relatively easy to set up and reproducible in terms of cell types.
You have to take into consideration three parameters : (a) date of gestation of the pregnant rat, if the age of embryo is critical ; (b) use of an adapted medium to dissect the tissues : the best medium is the L15 medium of LEIBOVITZ which allows to work at a constant pH although it does not contain sodium bicarbonate ; (c) dissection at +4°C. In our hand, this protocol allows to obtain reproducible cortical or hippocampal neuron suspension AND DRG.
Norbert and Phillippe, thanks for your replies!
I was going to use L15 for DRG Isolation anyways, perfect.
I guess the Cortex is easier to isolate in terms manual skills?
Hi Jörg,
I suggest you start with cortical neurons. Always use embryos with the sam age, E18 and use the culture at DIV 14 to DIV 18. We use silicone molds to standardize the cultures, make sure all cultures have the same pattern and to get more results/dish. We buy the molds from www.anandadevices.com. The molds can be used with cortical and DRG neurons. You can check our paper on electrophysiology here:
https://www.researchgate.net/publication/291333588_Rapid_Mechanically_Controlled_Rewiring_of_Neuronal_Circuits
Good luck!
Article Rapid Mechanically Controlled Rewiring of Neuronal Circuits
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