I have a very odd problem with my primary neuron cultures. They grow pretty well up to day 10-11 but after that they start to disintegrate.
The protocol is pretty straightforward:
I isolate the brain from embryonic mice and I triturate the brains with Trypsin, plate them in coated plates with PDL during 4 hours with DMEM supplemented with FBS. After that I change the medium and I add NBA.
The neurons grow very well up to day 11 after that they start disintegrating, it's not that they detach from the plate because I can still seeing the form of the neurons.
Any suggestions? I have tried different coating times, changing the media with different time intervals but the problem persist.
Hello Simon,
Suggestions:
- add laminin to PDL. It helps neurons to be maintained. For PDL, you have to wash it well with PBS (thrice) and once with strerile water.
- replace half of the culture medium every other day and add NGF (Nerve Growth Factor). I use Cf= 100 ng/mL for primary rat DRG neurons.
Hope this helps !
Zeina
Hi. Simon
you can change the half cultute medium every three days and add NAA.
Thank you so much!
Zeina Msheik and Yilin Pang one question, how is your coating protocol?
I will be aware.
Usually I use PLL 0.5 mg/mL (diluted in sterile distilled water) for 2h at room temperature (or you can leave it overnight at 4°C). Remove the PLL, wash 3 times with PBS, wash once with sterile distilled water and leave the surface to dry. I add a very little volume of the medium to the dish, and put it in the incubator for few minutes to warm. Then i add my cells.
If I want to use laminin , I use laminin C= 1.5 µg/mL. I add a volume 1:1 PLL:laminin. 2h at RT or O/N at 4°. Wash thrice with PBS. Add a very little volume of the medium to the dish, and warm it. Then i add my cells.
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