1. Keep 25-cm2 "T" flasks of cells in log phase of growth by subdividing when almost confluent. Freezing should only be carried out when cells are less than confluent and appear healthy.
2. Make sure that caps are tightened on flasks and then sharply tap to dislodge cells.
3. Pour off cell suspension into a sterile plastic universal container.
4. Pellet cells at 400g for 5 min.
5. Pour off medium, add 0.2 mL of 1% sodium azide solution and retain for assessment of antibody activity.
6. Tap the cell pellet and add 0.5 mL of cold freezing medium.
7. Aspirate with Pasteur pipet to resuspend pellet then transfer to a 2-mL cryogenic vial.
8. Cells must be frozen at approx 1°C/min and this can be achieved by putting the cryovials into an expanded polystyrene container with a wall thickness of 0.5 cm and then placing them in a -70°C freezer.
9. Transfer the cells to liquid nitrogen storage within 3 d of freezing at -70°C, they will remain viable for many years.
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