Upon receipt ampoules should be transferred to gaseous phase of liquid nitrogen if not to be used immediately. Do not use a -80^o freezer as an alternative.

Note: This is very important as it will minimise any loss of viability.

Resuscitation of Cells from Gaseous Phase Liquid Nitrogen.

1. Handle ampoules with care and wear laboratory coat, full protective face mask and gloves. Occasionally ampoules explode on warming. 
2. Leave ampoule at room temperature for approximately 1 minute and transfer to a 37^o C waterbath for 1-2 minutes until fully thawed. It is important to thaw quickly to minimise any damage to the cell membranes. Note: Do not totally immerse ampoule as this increases the risk of contamination.
3. Wipe ampoule with a tissue soaked in 70% alcohol prior to opening.
4. Slowly pipette the contents of the ampoule into a flask of pre-warmed medium in order to achieve the correct cell density specified on the enclosed cell line datasheet. A centrifugation step to remove cryoprotectant is not normally necessary (the centrifugation itself may damage the cells) and the DMSO eventually becomes diluted out during subsequent sub-culture. If centrifugation is required then this will specified on the data sheet.
5. Gas with 5% CO₂ if appropriate and incubate at the recommended temperature. Please refer to cell line datasheet enclosed.  Note: Some hybridomas may be slow to resuscitate.  Therefore start in 20% FBS and 10% hybridoma enhancement supplement (Sigma H8142) and DO NOT centrifuge to remove DMSO.