European Collection for Biomedical Research

Recommended handling of frozen cells

185

The ampoule(s) received from ECBR can be used immedlately or stored at a temperature below -80°C. preferably in liquid nitrogen vapor.

Thawing of the cells should be rapid. Remove the amoule from the dry ice or liquid nitrogen and place it directly into a water bath at 37-40°C with moderately vigorous agitation. As soon as the ice has melted (within 40-60 seconds), remove the ampoule from the water bath.

Quickly transfer the content of the ampoule in a sterile tube containing at least 10 volumes of culture medium (RPMI 1640 supplemented with 20 mM L-glutamine and 10% heat inactivated Foetal Calf Serum) and centrifuge the suspension at approximately 125 g for 10 minutes, discard the supernatant, resuspend the cells in 10 ml of culture medium. and transfer into a 50 ml culture flask.

Store the flask in a 5% C02, 37°C incubator. After 24 hours check the viability of the cells by dye exclusion and the cell count, and dilute them to a concentration of 2-3x105/ml.