this is my first time to teypsinize this cell line. I added 0.7 ml trypsin to 25 flask and observed from 2-8 minutes but most of cells did not detach. The trypsin works good as I used with other cell line same day. Shall I wait longer than 8 minute or increase trypsin time or what? Picture attached. Thanks.
What cells are you using? Did you wash the cells at least twice with PBS (containing no calcium or magnesium) before adding trypsin? Some cells stick very hard so you need to wash out serum, Ca and Mg for optimum trypsinization.
Hi Heba,
What concentration of trypsin did you use (0.05%; 0.25%)?
I suggest trying 0.25% trypsin with 1 mM EDTA at 37C for 5 - 10 min.
Hi Heba,
I understand that's a rat prostate cancer cell line, but have not worked with it. I suggest you contact someone growing these cells to see if they are normally difficult. I've seen trouble trypsinizing cells for these reasons. 1) cell lines that polarize and/or form tight junctions if left to differentiate or become too overgrown in the flask- example MDCK. They require 'stronger' trypsin/EDTA as John suggests such as 0.25% trysin, 2.21mM EDTA, and up to 20 minutes and/or double trypsin treatment. Some people passage difficult to detach cells by scraping instead of using trypsin. 2) Serum and Mg/Ca were not removed before trypsinizing, which you say is not the case. Washing with 1mM EDTA in PBS before trypsining, instead of just PBS, may help.
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