Prepare freezing mix (FM) in advance of the procedure. To do this, mix 10 mL of FBS with 8 mL of CM and 2 mL of dimethyl sulfoxide (DMSO) and store at 4°C (see Note 21).
1. Harvest the cells and supernatant from five 225-cm2 flasks 1 d after being seeded (see Note 22) and pool the resulting cell suspension.
2. Remove 2 mL of this suspension and add to a 25-cm2 flask. Supplement with 3 mL of fresh CM and place the flask in the incubator (see Note 23).
3. Centrifuge the remaining suspension at 300g for 10 min at 4°C. Retain the supernatant for antibody analysis. From this stage onwards all reagents should be used at 4°C. Recover the cell pellets, pool the cells, resuspend in 50 mL of CM, and wash by centrifugation at 300g as before.
4. Discard the wash supernatant and resuspend the cell pellet in 20 mL of fresh CM. Calculate the cell density by diluting 10 ^L of suspension in 90 ^L of nigrosin and counting the cells using a haemocytometer.
5. Centrifuge the cells as before then finally resuspend the pellet to a density of 5 x 106 viable cells/mL in FM and 1 mL added to each cryovial.
6. Place the vials were placed in a Nalgene freezing tub filled with iso-propanol then place in a -70°C freezer overnight (see Note 24).
7. Transfer the vials to LN2 for long-term storage (see Note 25).
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