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Harvest cells from a culture that is at or near peak density by centrifugation at 800 x g for 5 min.
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Adjust the concentration of cells to 2 x 106 - 2 x 107/mL in fresh medium.
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While cells are centrifuging prepare a 15% (v/v) solution of sterile DMSO in fresh medium.
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Mix the cell preparation and the 15% DMSO in equal portions. Thus, the final concentration will be 106 - 107 cells/mL and 7.5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution to the beginning of the freezing process should be no less than 5 min and no greater than 15 min.
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Dispense in 0.5 mL aliquots into 1.0 - 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
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Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.)
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The frozen preparations should be stored in either the vapor or liquid phase of a nitrogen refrigerator. Frozen preparations stored below -130°C are stabile indefinitely. Those stored at temperatures above -130°C are progressively less stabile as the storage temperature is elevated. Vials should not be stored above -55°C.
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To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial just to a level just above the surface of the frozen material. Do not agitate the vial.
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Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and add to a centrifuge tube containing 5 mL of ATCC medium 1747. Centrifuge at 300 x g for 5 min.
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Remove most of the supernatant (=cryoprotectant, which can inhibit growth) and then resuspend the pellet. Transfer the culture to a 16 x 125 mm screw-capped test tube containing 5 mL of ATCC medium 1747.
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Incubate on a 15° horizontal slant with the cap screwed on loosely (loosened one half turn) at 25°C under a 14 hour light (~50 µEinsteins/m2/s irradiance)/10 hour dark cycle.
