NS-O myeloma cells are normally kept stored cryogenically and so should be resuscitated 3 d before conducting the fusion.
1. Quickly thaw a frozen aliquot of NS-O cells either in a 37°C water bath or between the palms of the hands. Once the pellet has melted, add 1 mL of 37°C RPMI 1640 medium supplemented with 10% FBS and draw up into a Pasteur pipet. Transfer the cells to a 225-cm "T" flask and add 50 mL of RPMI 1640 medium containing 5% FBS and place in a tissue culture incubator 37°C/5% CO2.
2. Inspect the cells on d 2, they should be semi-confluent and adherent to the flask base. Increase the volume of RPMI 1640/5% FBS medium to 75 mL and return to the incubator.
3. Inspect the cells on d 3, they should be almost fully confluent on the flask base. Discard the medium, add 10 mL of cold sterile PBS containing 0.02% ethylene diamine tetraacetic acid and leave for a few minutes to allow the cells to detach. Split the cells equally between two flasks and add 75 mL of RPMI 1640 medium/5% FBS to each flask and return to the incubator.
4. On d 4, remove the medium from each flask and replaced with 20 mL of cold sterile PBS containing 0.02% ethylene diamine tetraacetic acid. After a few minutes the cells will detach from the flask surface. Harvest the cells by gently tapping the flask and pouring the cell suspension in to sterile universal containers. Wash the cells by cen-trifugation at 300g and resuspend in 10 mL of cold PBS.
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