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1. To harvest the Neospora culture, detach any remaining tissue culture cells (infected and uninfected) by scraping the surface of the flask with a cell scraper.
2. Transfer the cell suspension (including parasites) to 15 mL plastic centrifuge tubes. Centrifuge at 1300 x g for 10 min.
3. Remove all but 0.5 mL of the supernatant from each tube, resuspend the cell pellets, and pool them to a single tube.
4. Pass the resulting cell suspension through a syringe equipped with a 27 gauge 1/2 in needle to break up any remaining cells. Adjust the parasite concentration to 2.0 - 4.0 x 10⁷ cells/mL with fresh medium or PBS.
   NOTE: If the concentration of parasites is too low, centrifuge at 1300 x g for 10 min and resuspend in the volume of fresh medium or PBS required to yield the desired concentration.
5. Prepare a cryoprotective solution containing 20% (v/v) DMSO and 6% (v/v) HIFBS in fresh medium or PBS.
6. Mix the cell preparation and cryoprotective solution in equal portions. The final concentration will be 1.0 - 2.0 x 10⁷ cells/mL, 10% DMSO, and 3% HIFBS. 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.
   NOTE: To prevent culture contamination, penicillin-streptomycin solution (ATCC^® 30-2300) may be added to a final concentration of 50 to 100 I.U./mL penicillin and 50 to 100 µg/mL streptomycin.
7. Dispense in 0.5 mL aliquots to 1.0-2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
8. Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C plunge ampules 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.)
9. Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator.
10. 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.
11. Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of CCL-81™ cells and 10 mL ATCC^® 30-2003 with 3% (v/v) HIFBS.
12. Outgas the flask for 10 seconds with a 95% air, 5% CO₂ gas mixture.
13. Incubate in a 35°-37°C CO₂ incubator with the cap screwed on tightly.

Dubey JP, et al. Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. J. Am. Vet. Med. Assoc. 193: 1259-1263, 1988. PubMed: 3144521