canine prostatic cell isolation

[sepideh]

Hello all,

I am trying to isolate canine prostatic epithelial cells using an enzymatic method. I am successful in doing that to some degree. Originally the cells adhere to the collagen coated surface within 24-36 hours and they start proliferating but then they die shortly following the first pass.

I was wondering if any of you have had a similar experience or might have some suggestion regarding what might be wrong. I greatly appreciate any help that I can get.

Regards,

Sepideh

[danfive]

Are you using trypsin or scraping the cells before passing? What media are you growing them in?

[sepideh]

I was originally using .25% trypsin, for 2-3 minutes. But last time that I isolated cells I switched to .025% trypsin for about 10 minutes, that is what ATCC recommends for human prostatic epithelial cell lines.

[sepideh]

Sorry. If I am not mistaken, they recommend exposure to trypsin (.025%) for 5-8 minutes, but I had to keep them for 10-11 minutes for the cells to come off.
I grow them in a media made by Cambrex which is specialized for human prostate epithelial cells, but I have been told would work for canine ones as well.
Also thank you for your time; I am really clueless in what might be wrong.

Thanks,

Sepideh