Good day to all,
We are extracting and cultivating cortical neurons from P0-P2 rat pups. But they seem to stay depolarized in culture and we don't understand why. Morhologically they look good, healthy but they do not show spontaneous activity in MEA measurement or under patch clamp.
Could anybody give a hint what we are doing wrong?
for reference:
- P0-P2 rats, cryoanesthesia and decapitation, cortical dissection, cut in tissue chopper (100µm x 100 µm)
- digestion in accutase (20 min at 37°C)
- seed after membrane filtering (45-75 µm)
- seeding medium: DMEM with 10% FBS + Pen/Strep (only 3 hours for attachment)
- feeding medium: Neurobasal A with B-27, Glutamax (1 mM), Normocin
- medium change: 2x a week, only half is changed, no aspiration
--> Patch at 10 days: all depolarized (~ -30 mV)
--> Patch at 16 days: all depolarized (~ -30mV)
Hii Muammer Üçal ,
Have you or somebody else has done the cortical neuronal culture earlier?
If No, then humorous factors could be one of the probable reason that led to depolarization of neurons. Since our culture media consist of minimal compounds that we have established earlier, In contrary to that our circulatory system carries thousands of the factors with varying level their synchronization affect the development of in vivo system.
if you think the same reason, I suggest to use the fetal/pups serum of rat that may help you to get good results.
Regards
Dear Muammar,
the most likely explanation is an elevated K+-concentration in the culture medium. Please check this in the Neurobasal medium and in your recording solutions. Is there a difference in the magnitude of depolarization between Neurobasal and recording solution? This could give you a hint. Also, the Glutamax and normocin stock solution might contain (by accident) elevated K+.
Best wishes, Volkmar
Dear Muammar,
I also suspect that there is a problem with the medium. Maybe you can consider to feed the cultures with BrainPhys™ Neuronal Culture Medium , whith which we have some good experience, or similar, which provides a more physiologcial envirroment for the neurons. Also, in our hands cultures grow and develop best if change the medium as little as possible and completely leave the cultures untouched the first week. From Div 7 on you should then definetly see some activity with the MEA.
Best, Anne
Dear Muammar,
If - medium change: 2x a week is not enough to maintain a good energy performance of neuronal mitochondria for 10 days, then an increase in the concentration of calcium in the cytosol is possible up to a critical one.
The easiest way to test mitochondrial status on a 10 day old cell homogenate of ROS production is by adding respiration substrates for complex one and complex two.
Hi, I guess what you see is due to the presence of too many glial cells in your culture. I would recommend that after 5 to 7 days, add 1,5 mM of 1-b-D-Arabinofuranosyl cytosine (AraC) (SIGMA #C1768) to stop astrocyte proliferation.
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