Hi all,
I am working on both primary hippocampal mouse neurons and i3Neurons to isolate axonal material. I plate them in microfluidic devices in house made (like any other you can find on the literature). Two chambers, an axonal and a somal separated by microgrooves.
For the primary, I plate 500.000 cells on memHorse, and the next day I change to Neurobasal. Then on DIV3, I start a gradient of BDNF (20 ng/mL on somal side and 50 ng/mL on axonal side). I keep this gradient for 3 days and then I keep the cells with 10 ng/mL of BDNF in both chambers. I always use conditioned Neurobasal from glial cells diluted 1:5 with fresh neurobasal on top of the 10 ng/mL BDNF. I refresh the medium every 3 days (with this conditioned medium diluted 1:5). My cells look fine during the first 5 days, then they start to clump a lot (this happens 80% of the times). I have done lysosomal trafficking recordings on them and they seem healthy, but there is something I do not control and I was wondering if any of you could have an answer.
I coat with PLL in borate buffer over night.
It is not a problem of the cells, because in paralel I also plate on coverslips and they are fine.
My thoughts are:
Maybe the BDNF concentration is not okay (I read that too much can also induce a lot of growth, what may be why they are pushed against each other, because the neurites grow so much against the walls that the cell bodies get together).
Maybe the conditioned medium and BDNF at the same time is too much and I should try only conditioned medium or only BDNF.
Maybe the cell density is not okay, although so far I have tried 500.000, 300.000 and 150.000 and it doesnt seem to be the matter...
Thank you very much! Any tip would be incredibly welcome :)
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