Toxoplasma gondii (Nicolle and Manceaux) Nicolle and Manceaux (ATCC® PRA-319)

Organism: Toxoplasma gondii (Nicolle and Manceaux) Nicolle and Manceaux  /  Depositor: V Carruthers

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Strain Designations RHΔku80Δhxg
Application
Food and waterborne pathogen research
Opportunistic pathogen research
Biosafety Level 2
Isolation
Double knockout generated in a RH strain background that lacks the ku80 and hxgprt genes
Product Format frozen
Type Strain no
Medium ATCC® Medium 2222: Cell Cultivation Medium for Parasites
Growth Conditions
Temperature: 35-37.0°C
Growth condition: Grown in human foreskin fibroblasts ATCC CRL-1634
Cryopreservation

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 cell suspensions (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 - 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 parasite preparation and 15% (v/v) DMSO in equal portions.  The final concentration will be 1.0 - 2.0 x 107 cells/ml and 7.5% DMSO.  The time from the mixing of the cell 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 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 thawing.

10.          Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC CRL-1634 cells and 10 ml ATCC 30-2002 with 10% (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 V Carruthers
References

Huynh M, Carruthers VB. Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot. Cell. 8: 530-539, 2009. PubMed: 19218426

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