I am isolating the bone marrow cells from mice and culture those cells. Every time when I try to subculture the cells, the cells do not detach. I tried using 0.05% Trypsin in 1X EDTA and also 0.25% Trypsin in 1X EDTA. In both condition cells don't detach. I even tried from 2-10 mins still cells too much adherent. I wash with DPBS with out calcium and magnesium chloride twice prior to enzyme treatment. I am using both rat as well mice for bone marrow stromal cells. But the problem is only with mice cells. Please give me an idea.
You say it works for the rat cells but not mouse cells. 0.25% is correct.
1. Is the 0.25% trypsin fresh? Did you try newly thawed Tryspin? because the trypsinization step is pretty standard for mouse stromal also..
you can cells detach with 0.25% trypsin/0.02% EDTA for 5 minutes at 37°C. The residual cells which were not detached within 5 minutes were collected by gentle scraping , it was transferred to other culture dish and add new DMEM medium . after that , incubate cells at 37°C, 5% CO2 and 90% humidity .
I would like to thank you all very much. And do anyone know the idea about why mesenchymal stem cells get hazzy after changing the concentration of serum in the media. Even the cells get so flat type.
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