1. Harvest cells from a culture which is at or near peak density by centrifugation at ~800 x g for 5 min.
2. Adjust concentration of cells to 0.5–1.0 x 10⁷/mL in fresh growth medium. xa0If the concentration is too low, centrifuge at ~800 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.
3. While cells are centrifuging, prepare a 20% (v/v) solution of sterile glycerol in fresh growth medium.xa0
4. Mix the cell preparation and the glycerol solution in equal portions. The final concentration will be 2.5-5 x 10⁶ cells/mL in 10% glycerol. The time from the mixing of the cell preparation and glycerol stock solution before the freezing process is begun should be no less than 15 min and no more than 30 min.
5. Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryovials.
6. Place the ampules in a Nalgene 1°C freezing apparatus.xa0 Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen.xa0 (The cooling rate in this apparatus is approximately -1°C/min.)xa0 If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion.xa0 At -40°C, plunge ampules into liquid nitrogen.
7. Store in either the vapor or liquid phase of a nitrogen refrigerator.
8. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.xa0 Do not agitate the ampule.xa0 Do not leave ampule in water bath after it is thawed.
9. Remove the vial from the water bath immediately after thawing.xa0 Aseptically transfer the contents of the ampule into 10 mL of fresh growth medium.xa0
10. Incubate at 37°C under 5% CO₂ atmosphere.
11. Maintain as described above.xa0

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