Upon receipt ampoules should be transferred to gaseous phase of liquid nitrogen if not to be used immediately. Do not use a -80o 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. - Handle ampoules with care and wear laboratory coat, full protective face mask and gloves. Occasionally ampoules explode on warming.
- Leave ampoule at room temperature for approximately 1 minute and transfer to a 37o 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.
- Wipe ampoule with a tissue soaked in 70% alcohol prior to opening.
- 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.
- 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.
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