Hi,
I am trying to standardize the culture of Zebrafish spinal cord ependymal cells. I dissect the spinal cord. Dissociate the spinal cord by trypsinization and sort the GFP labelled ependymal cells using FACS. Then, I plate these cells in Poly-L-Lysine coated plates containing DMEM/F12 or N2B27 medium and incubate them in an incubator at 28 degrees. Ependymal cells attached to the plate and I also checked the GFP expression. However, the cells are not dividing. They are suppose to divide a few times. How can I troubleshoot this problem.
Thanks,
Ridim
I am not adding any growth factors. My media contains DMEM/F12 + 20% FBS + Glutamine and penstrep as antibiotic. What growth factors do you recommend? And does zebrafish ependymal cells require 5% CO2 during incubation in the incubator?
20 % is a lot of serum. You could be getting an inhibitory effect. The basal medium determines whether you need CO2. L-15 is often used for Zebrafish and does not use CO2. I would try that first.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128108/
I have ordered L-15 media. I will definitely try that. Thanks a lot for your suggestions.
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