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Gentamicin for isolation - (Aug/15/2006 )


I have been trying to isolate limbal epithelial cells, but I always get some fibroblasts in the culture which end up taking over.
I have been advised to use Gentamicin, which has a larger effect on the fibroblasts than the epithelial cells, but I cannot figure out if I must add it during the isolation with Dispase II or during the culturing. Anyone know when it can be used and in what concentrations?
Also can I use G418 Sulphate? Is it the same?



We have used differential plating in the past when isolating primary rat smooth muscle cells. Once you obtain your epithelial cell suspension, incubate it for 10 minutes to allow the fibroblasts to stick to the plastic. Wash the non adherent cells off and do this again x2. In this way we have reduced any fibroblast contamination to next to nothing (determined by immunostaining of alpha actin).
I have not heard of the Gentamycin protocol, and I would be worried of using any compound that would effect the cells directly.


Gentamycin is an antibiotic that kills bacteria, but not eukaryotic cells. (colleagues of mnie use it instead of pen/strep).

G418 is geneticin, some topics on this forum concern G418, so you can find more info about it.