Cell Freezing and Thawing Protocol

Thaw cell culture freezing medium, mix well by gently swirling bottle. Keep medium on ice during use.
Cells to be frozen should be in late log phase growth.
Monolayers will need to be dissociated. After dissociation, cells are resuspended in complete growth medium and counted to determine viability and number.
Gently pellet the cells. Remove the medium above the pellet.
Resuspend the cells in cell culture freezing medium at a concentration of ~5 x10⁶cells/mL to 1 x 10⁷ cells/mL. Hybridoma cells should be resuspended at a concentration of ~1 x10⁷cells/mL to 1 x10⁸ cells/mL. Freeze 1 mL of cells/vial. After the cells have been resuspended and aliquoted into appropriate cryogenic storage vials, they can be placed on dry ice and your normal freeze down procedure should be started within five minutes.
Cells can be stored at a minimum temperature of –80°C, but for long term storage the cells should be stored in ultra-low temperature freezers (–150°C) or in liquid nitrogen (–196°C).
Thawing of cryopreserved cells should be as follows:

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