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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/2.5/2
TITLE: RESUSCITATION OF FROZEN CELL LINES
This protocol describes the resuscitation of cells stored in either liquid or vapour phase of liquid nitrogen.
- Obtain the location of cell line from the relevant database.
- Wear protective clothing, i.e. lab coat, protective face mask and gloves.
- Remove the required ampoule(s) from liquid nitrogen tank and place ampoule in a screw top container within a transport tin and transport to the laboratory.
- Still wearing protective clothing place ampoule in an ampoule rack and into a 37°C ± 1°C water bath until thawed (usually 3 to 5 minutes). Do not submerge the ampoule, ensure the cap is above the water level.
- Transfer to Class II or Class III cabinet as appropriate and open the ampoule using an alcohol drenched tissue.
- Slowly dilute out the DMSO with warmed cell culture media and take up the cells into approximately 10ml of media.
- Centrifuge the cells at 150g for approximately 5 minutes at 15 to 25°C.
- Pour off the supernatant and resuspend the pellet of cells in fresh medium.
- Seed tissue culture flasks with the required seeding density into pre-warmed media and incubate at the required temperature according to the cell line data sheet specific to the cell line.
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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