Dispase - Specific protocole for the use of Dispase (Jun/11/2002 )
I need to separate fibroblasts from epithelial cells. I know that dispase, neutral protease is used for that. Can anyone give me a specific protocol for the use of this protease ?
I used 0.25% trypsin to seperate fibroblasts from epithilial cells. And it really did. Why not use trypsin?
I thought that dispase, from what I read, was effective epithelial cells, and that trypsin is too strong (it is active on both). Can you tell me when you make the selection (after 24 or 48 hrs) after the initial plating of those primary cells ? How do you make sure that only epithelial cells are sensitive to this treatment ?
What I did is primary culture of lung tumor cells. And I applied cold digestion method. About 72hrs after plating cells, I applied 2ml 0.25% trypsin to the flask and observed under inverted microscope. Stop digestion when found the fibroblasts began to shrink. Then added medium (including FBS), shook gently and poured out.
Usually I got above 90% of epithelia.
And I have thought to apply dispase to primary culture,but GIBCO company had stopped providing it.
Thank you !