Neospora caninum Dubey et al. (ATCC® 50845)

Organism: Neospora caninum Dubey et al.  /  Depositor: LD Sibley

Permits and Restrictions

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Strain Designations Nc-LIV [NC-Liverpool]
Application
It produces both tachyzoites and bradyzoites in vitro and in vivo.
Biosafety Level 2
Isolation 5-week-old Boxer puppy, Liverpool, England
Product Format frozen
Storage Conditions Frozen Cultures:
-70°C for 1 week; liquid N2 vapor for long term storage

Freeze-dried Cultures:
2-8°C

Live Cultures:
See Protocols section for handling information
Type Strain no
Comments
This organism is an obligate intracellular parasite that can infect a variety of mammalian cell lines.
It produces both tachyzoites and bradyzoites in vitro and in vivo.
The strain is negative for mycoplasma contamination.
Growth Conditions
Temperature: 35°C
Culture System: ATCC® CCL-81™ (African green monkey kidney cells)
Cryopreservation Harvest and Preservation
  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.
Name of Depositor LD Sibley
Chain of Custody
ATCC <-- LD Sibley <-- A.J. Trees
References

Barber JS, et al. Characterization of the first European isolate of Neospora caninum (Dubey, Carpenter, Speer, Topper and Uggla). Parasitology 111: 563-568, 1995. PubMed: 8559588

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation