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Sarcocystis neurona

PRA-420

Product category
Protists
Product type
Parasitic protozoan
Classification
Sarcocystidae
Strain designation
SN3.E1
Genome sequenced strain
Yes
Isolation source
Spinal cord homogenate from 2-yr-old thoroughbred filly
Geographical isolation
Panama
Applications
Agricultural research
Product format
Frozen
Storage conditions
-80°C or colder for 1 week, vapor phase of liquid nitrogen for long-term storage
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Documentation

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

BT

CRL-1390

Detailed product information

Characteristics

Comments
Causative agent of equine protozoal myeloencephalitis (EPM)

Handling information

Host
Instruction for complete medium
Media: Advanced Minimum Essential medium (MEM) (Gibco Cat # 12492-013) supplemented with 4% fetal bovine serum (ATCC® cat. 30-2020).

Alternative media: ATCC® 30-2002 [Dulbecco's
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure

Cell Line Maintenance

  1. To establish a cell culture from the frozen state, place an ampule of ATCC® CRL-1390™ in a water bath set at 35°C for 2-3 min. Immerse the vial just sufficient to cover the frozen material. Do not agitate the vial.
  2. Immediately after thawing, aseptically remove the contents of the ampule and inoculate into 10.0 mL of fresh Advanced MEM with 4% (v/v) heat-inactivated fetal bovine serum (HIFBS)* in a T-25 tissue culture flask.
  3. Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.
  4. Incubate in a 37°C CO2 incubator with the caps screwed on tightly.
  5. Change the medium 1-2 times per week.  
    *Fetal bovine serum is available from ATCC (catalog number 30-2020). Serum is heat-inactivated by exposure to 56°C for 30 minutes. This treatment will inactivate proteins of the complement pathway. Remove the serum from the refrigerator and aseptically distribute in 100 mL aliquots to sterile 125 mL screw-capped bottles. Immerse bottles in a 35°C water bath for 5 minutes. Do not directly transfer bottles from the refrigerator to 56°C. Transfer the bottles to a 56°C water bath and begin timing for 30 minutes. To avoid contamination, do not allow the level of the water in the bath to come in contact with the lip of the screw cap. It is best to leave one inch between the serum level in the bottle and the lip of the cap and to fill the water bath to a level just slightly above the level of the serum. To assure even heating of the serum, swirl the bottle(s) every ten minutes. Note: Some suppliers provide serum already heat-inactivated.

 

Transferring the Cell Line

  1. When the cell line forms a confluent layer, remove all the medium and replace it with 2 mL of 0.25% (w/v) trypsin dissolved in Hank's Balanced Salt Solution.
  2. Gently distribute the trypsin over the monolayer, remove the trypsin, and place the flask at 37°C for 5 min.
  3. Add 2 mL of Advanced MEM with 4% (v/v) HIFBS in a T-25 tissue culture flask and detach any cells still adherent by alternately aspirating the medium into a pipette and discharging the contents over the monolayer.
  4. Distribute the cell suspension in 0.5 mL aliquots to four T-25 flasks containing 10 mL of fresh culture media.
  5. Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.
  6. Incubate in a 37°C CO2 incubator with the caps screwed on tightly.

 

Storage and Culture Initiation
Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen.  If liquid nitrogen storage facilities are not available, frozen ampoules may be stored at or below -70°C for approximately one week.  Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C).  Storage of frozen material at this temperature will result in the death of the culture.
  1. 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 it is thawed.
  2. Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC® CRL-1390™ cells and 10 mL of Advanced MEM with 4% (v/v) HIFBS.
  3. Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.
  4. Incubate in a 37°C CO2 incubator with the caps screwed on tightly. Observe the culture daily under an inverted microscope for the presence of schizont and merozoite stages of Sarcocystis.

Culture maintenance
  1. Remove the medium from a fresh confluent monolayer of ATCC® CRL-1390™ cells in a T-25 tissue culture flask and replace it with 10 mL of Advanced MEM with 4% (v/v) HIFBS.
  2. To transfer the Sarcocystis culture, remove the medium containing free merozoites and centrifuge at 1300 x g for 10 min.
  3. Remove the supernatant and resuspend the parasite pellet in 1 mL of culture medium.  Transfer the resuspended pellet to the fresh flask of CRL-1390™ cells.
  4. Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.
  5. Incubate in a 37°C CO2 incubator with the caps screwed on tightly.

 

Cryopreservation
  1. Harvest the Sarcocystis 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 infected and uninfected tissue culture cells by scraping the inner surface of the flask with a cell scraper. Pass the resulting cell suspension through a syringe equipped with a 27-gauge ½-inch 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 supernatants to new 15 mL plastic centrifuge tubes.  Centrifuge at 1300 x g for 10 min.
  4. Pool the parasite pellets and adjust the concentration to ~2.0 x 107 merozoites/mL with fresh Advanced MEM.
    *If the concentration is too low, centrifuge at 1300 x g for 10 min and resuspend in the volume of Advanced MEM required to yield the desired parasite concentration.
  5. Mix equal volumes of parasite suspension and fresh medium containing 20% DMSO and 50% HIFBS to yield a final concentration of ~1 x 107 merozoites/mL in 10% DMSO, 25% HIFBS.  The time from the mixing of the parasite preparation and the cryoprotective solution before the freezing process begins 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 cryovials.
  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 thawing.
  10. Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC® CRL-1390™ cells and 10 mL of Advanced MEM with 4% (v/v) HIFBS.
  11. Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.
  12. Incubate in a 35-37°C CO2 incubator with the caps screwed on tightly.

History

Depositors
DK Howe
Type of isolate
Animal
Year of origin
1991
Cross references
GenBank JAQE00000000 Sarcocystis neurona strain SN3 clone E1, whole genome shotgun sequencing project

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

United States Veterinary Permit for Importation and Transportation of Controlled Materials and Organisms and Vectors

For every order of this item, you must provide a valid Permit for Importation and Transportation of Controlled Materials and Organisms and Vectors (VS Form 16-6A) obtained from the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service. We cannot ship this item until we receive this permit.

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

Frequently Asked Questions

References

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