Primary culture - keratinocytes - how do I know? (Nov/14/2005 )

Hi,

I am trying to culture canine keratinocytes. I have some cell growth but how do I know for certain that the cells are keratinocytes? Is there a definitive test I can do?

Thanks

do they look like these?

http://www.medscape.com/content/2000/00/40...205.03.fig2.jpg

http://www.amaxa.com/uploads/pics/keratino...light-24h_W.jpg

http://www.cell-systems.com/images/2BB3-P75.gif


they have a 'cobblestone' appearance under the inverted scope that is quite a distinctive morphology

I think perhaps there are is a panel of tests you could use to confirm, but I do not know what that would be. perhaps you could go to the literature?

thanks,

they don't really look like the 1at or 3rd pic but maybe more like the second one. I have been looking at the lit to find a method but not doing very well so far!

how/where did you get the cells? did you isolate them from an epidermal graft or something, or get them from someone else?

we buy primary human skin keratinocytes from a company that sends a little picture of how they are supposed to look. we maintain them as directed and monitor morphology and growth habits to be sure that the cultures continue to be keratinocytes.

I assume you used a standard isolation protocol to separate the layers (that whole dispase step)? then there should be two options in your dish at this point, assuming a nice clean culture: fibroblasts, and keratinocytes, right? they look quite different and if the cells you got look like the pix I linked to that one post, you should be good to go. we use epidermal keratinocytes, but I think pretty much any old keratinocyte looks pretty similar in the dish, according to what I have seen in the literature

http://www.cascadebio.com/pdfdocs/APF%20Ke...re%20042903.pdf

if you look at the latter half of this, you may find some helpful tests. I don't know how similar dog keratinocytes are to humans, but this might help you get started??