The curling of 3T3-L1 seems to be a common problem, and after reading many solutions, I still am encountering difficulties.
Here is the problem. My cells proliferate well until changed to differentiation medium. This is where the problem happens. The medium is replaced with short-term differentiation medium, containing DMX, IBMX, 10% FBS and insuline from bovine pancreas, which has been heated to 37 C and pH adjusted to 7.2. After 2 days in the medium, the cells appear fine, no curling has happened, but the medium has now turned yellow! When changing the media to long term differentiation, containing only FBS and insuline, I can litteraly see the cells curling before my eyes!
Here is what I have tried to remediate the problem:
-Warmed the medium to 37 before using.
- Used a 1ml pipette instead of succession so as to not disturb the cells during the change.
- Added the long term medium slowly with the pipette pressed against the edge of the well wall at 90 degree angle (or almost)
- With an initial 0.5 ml in each well (24 well plates) I remove 375 ul and add 475 ul (compensating for evaporation)
I still get curling!
Any ideas or solution?