I am culturing mouse fibroblast cell in 10% FBS medium with 1% PSG and 0.1% Amp B. What is the condition for this cell to freeze in liquid nitrogen?
Hi,
Our standard procedure for freezing fibroblasts is
1) Put ~ 1 million cells per cryotube into 1 mL of 10% DMSO + 90% FBS
2) Put tubes into a styrofoam container with a wall thickness of ~ 1 cm. You may need to do-it-yourself. Polyurethane insulating plates work similarly well and are available at hardware stores.
3) Transfer container to a -80°C freezer and leave it overnight. The container serves to achieve a freezing rate of ~ -1°C/min.
4) The next day transfer the tubes to liquid nitrogen. If you wait too long (> 1 week) viability will decrease significantly
Best wishes, Ernst
Freezing MEFs, particularly inactivated fibroblasts, is a great convenience.Cells are frozen slowly in medium containing 10% (v/v) dimethyl sulfoxide and thawed rapidly
Check out the full protocol here:
http://www.boneandcancer.org/MOLab%20protocols%20since%2011-2005/E20-mouse%20embryonic%20fibroblast%20(MEFs)%20isolation.pdf
I hope it will be helpful.
All the best
You really do not need 90% FBS in your cryopreservation medium. 20% is more than enough and the remaining 70% can be much cheaper DMEM.
Cells should be stored in liquid nitrogen. Do NOT store cells at -80°C. The cells are
extremely temperature-sensitive and should be transferred to liquid nitrogen immediately upon arrival. Cells should be transported on dry ice or
in a liquid nitrogen container. When transporting the cells on dry ice make sure that the vials are completely covered.
The recommended media for the MEF cells is DMEM high glucose containing 10% FBS.
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