Removing cells from liquid nitrogen
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- Put cryovial straight from storage and float in the 37� water bath- caution should be taken as on rare occasions vials can explode when heated up due to trapped liquid nitrogen.
- In a hood, using sterile technique, transfer 10mls of prewarmed FCS supplemented media into sterile centrifuge tubes.
- As soon as cells are warm take the vial from the water bath and clean the outside thoroughly with 70% ethanol.
- Pipette cells from the cryovial and slowly drop by drop transfer to the prepared 10mls of media.
- Spin immediately at 1000rpm for 5 mins, low brake in order to pellet the cells.
- Immediately remove the media from pellet.
- Resuspend cells in a fresh 10mls of media and transfer to a flask and grow in incubator.
- The following day take off media, wash with warm sterile PBS and add more media, leave to incubate for a further 24 hours.
- Check confluence of cells under the microscope and split by trypsination if necessary.
