After dissociating the mouse cerebellum, I can only see a lot of black staffs at the bottom of wells under optical microscope, but not the neuron cells such as granule cells. Is it because we can not see claws of neuron cells or the dissociation process went wrong?
Without further details, it is difficult to guess what the problem might be. In general, dissociation of cerebella froim P1 and P2 mice is a straightforward protocol that should yield a large number of viable neurons - mainly granular cells.
Following the dissociation it will take several hours for the neurons to adhere to the bottom of the well. Only then will you be able to easily find them using a phase-contrast microscope. While the cells will give clear contrast, they will not have any obvious 'claws'. But your 'black stuff' sounds as if you might not have an viable cells in the well at all.
How long have you had the cells in culture for when you see this debris? Have you tried culturing a cell line in parallel to verify that you techniques and equipment are OK? Which protocol are you using? Have you had an experienced colleague walk you through it, or did you go 'from book to bench'?
How often have you tried the protocol? It is critical that the preparation goes smoothly. When I was culturing cerebellar neurons, my first prep had very few live neurons. But I saw a major increase in the number of live cells from prep to prep. In the end, it really is a matter of practice.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Anatomy
- Tissue