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How to freeze and thaw
Freezing procedure.
Cells are kept in culture until they reach a confluence between 80-90%. Cell suspensions with viabilities of ≥75% are counted. Typically 2 x 10E6 cells are spun down at 350xg the cell pellets are re-suspended in 1ml of ice cold FILOCETHplus and transferred into a cryo-vial Samples are kept on ice for 10 min. The actual freezing process from 0°C to -80°C is by placing the samples in a polystyrene insolated box in a -80°C freezer. After ≥3 h but not later than after 3 days, samples can be transferred to the liquid nitrogen tank for long-term storage.
Thawing procedure
The cryo-vials are placed in a 37°C water bath for exactly 2 min (a little ice is still present in the tube). The cell suspension is diluted into 10 ml of fresh culture medium and centrifuged at 350xg for 5 min. The cell pellet is re-suspended in 6 ml of pre-warmed culture medium and plated into a 6-well plate. After 24 hrs the cells are checked visually and transferred in an appropriate culture vessel if needed.
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