Removing cells from liquid nitrogen

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.