I am doing cerebellar organotypic slice culture from mice p8 and would like to know if there is a way of making sure the slices are happy and alive?
I would also like to know if it is normal to see migration of cells (probably microglia??) outside the slice boundaries?
Thanks,
Orli
Several ways to assess their health, depending on what your end point measure is. The simplest would be to simply gauge that they maintain some semblance of their native morphology - I have no experience with cerebellar slices, but certainly hippocampal ones should look like they look in schematic figures for at least a couple of weeks in vitro.
If you or a neighbouring lab has access to electrophysiology set-ups, you can place them under DIC optics, and should be able to visualize the neurons clearly; you can go further by recording some functional electrical activity.
If you're interested in morphology, you should be able to find a way to label a subset of the cells - a transgenic mouse with fluorescent cells would be the simplest, but can also use lipohilic dyes like DiI. Can also simply label nuclei with Propidium Iodide, where labelling should be minimal in a good healthy slice (perhaps compare to a slice that you induce toxicity in).
In answer to your last question, yes it is normal for there to be cells outside the original boundary of the tissue you placed on the membrane - these cultured slices "thin out" with time, and there is glial migration.
I believe electrophysiological recording will assure that your neurons are alive and since they are by far more sensitive than glial cells, this would be enough.
-Marilena-
It is simple and quick to confirm the viability of your tissue during the culture.
Similar to organotypic hippocampal slice culture, few days after the mechanic slicing you will be able to see huge amount of microglia appear, the tissue will get thickened and swollen and under microscope it will be dim because it is too thick to allow the light to pass through. At this stage the brain slice is recovering from the mechanic damage and its viability is unstable.
Gradually, if the tissue recovers well, microglia will disappear (you might still see some), dead cells will be scavanged and cleared out, the slice will become thinner and you can find bright and intact structure under microscope. This proccess normally takes 14 days after slicing.
However, you can still try to detect the viability through PI staining, but be careful because PI could be toxic to the tissue.
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