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1. Harvest the culture by gently agitating the contents of each flask.  Transfer all but approximately 1 mL of the culture medium to 15 mL plastic centrifuge tubes. Detach the remaining tissue culture cells (infected and uninfected) by scraping the surface of the flask with a cell scraper. Pass the resulting cell suspension through a syringe equipped with a 27 gauge 1/2 in needle and pool this suspension with the culture fluid.
2. Spin the cell suspensions at approximately 50 x g for 3 min, to remove the cellular debris.
3. Transfer the spore suspensions (supernatants) to new 15 mL plastic centrifuge tubes.  Centrifuge at 1300 x g for 10 min.
4.  Pool the spore pellets and adjust the concentration to 2.0 - 4.0 x 10⁷ cells/mL with a fresh solution of Hank's Balanced Salt Solution.
   *If the concentration is too low centrifuge at 1300 x g for 10 min and resuspend in the volume of Hank's Balanced Salt Solution required to yield the desired concentration.
5. Mix the spore preparation and 20% (v/v) DMSO in equal portions.  The final concentration will be 1.0 - 2.0 x 10⁷ cells/mL and 10% DMSO.  The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.
6. Dispense in 0.5 mL aliquots to 1.0-2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. 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.)  
8. Store in either the vapor or liquid phase of a nitrogen refrigerator.
9. 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.  Do not agitate the ampule.  Do not leave ampule in water bath after thawed.
10. Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC^® CCL-81™ cells and 10 mL ATCC^® 30-2003 with 3% (v/v) HIFBS.
11. Outgas the flask for 10 seconds with a 95% air, 5% CO₂ gas mixture.
12. Incubate in a 35°C CO₂ incubator with the caps screwed on tightly.

Dubey JP, et al. Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med. Assoc. 192: 1269-1285, 1988. PubMed: 3391851

Dubey JP, Lindsay DS. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67: 1-59, 1996. PubMed: 9011014

Dubey JP, et al. Redescription of Neospora caninum and its differentiation from related coccidia. Int. J. Parasitol. 32: 929-946, 2002. PubMed: 12076623