Hello!
I have been attempting to create cortical primary cell cutlure from P2 C57Bl/6 pups. Nevertheless I repeatedly end up with massive amounts of debris. The procedure I follow is the standard one and for the dissociation I use the NeuroCult™ Enzymatic Dissociation Kit. Anybody has some input or any troubleshooting on this issue? Thank you very much in advance!
A P2 mouse is too old for primary neurons, it is usually used to prepare astrocytes which are plentiful at that stage. You must use a E14-E16 where the population is mostly neurons and the cells are easier to dissociate. You might have to practice a little as the tissue is much softer and delicate than a P2.
Hello Elizabeth and thanks for the answer! All my past experience was with embryos but unfortunately now I have to do it postnatally.
I agree with Elizabeth. Right after birth (p0-p1) there is a massive wave of gliogenesis in the brain and glia cells mostly die in selective neuronal culture, hence your debris. If you have enough surviving neuron in the culture you can just wash off the debris but I don't think you can adapt your protocol to get much more surviving neurons from a postnatal brain. BTW, if you are making neurosphere culture the neurosphere will mostly be gliogenic too. I wonder why you have to do your culture postnatally.
Thank you very much Xiwei! I get your point. I was wondering whether the debris was indeed glial components or cellular parts, but i tend to incline to the first hypothesis. As for your question it has to do with restrains from the part of our animal facility.
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