Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

1.xa0xa0 Harvest Trypanosoma cultures when emergent parasites (trypomastigote stage) have reached or are near peak density in the liquid column. Gently invert the Trypanosoma culture flasks to suspend parasites in the liquid medium.

2.xa0xa0 Transfer the cell suspension (including parasites) to 15 ml plastic centrifuge tubes. Centrifuge at 1300 x g for 10 min.

3.xa0xa0 Remove all but 0.5 ml of the supernatant from each tube, resuspend the cell pellets, and pool them to a single tube.

4.xa0xa0 Adjust the parasite concentration to 2.0 - 4.0 x 10⁷ cells/ml with fresh medium or PBS.

xa0xa0xa0xa0xa0 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.xa0xa0 Prepare a cryoprotective solution containing 10% (v/v) DMSO in fresh medium or PBS.

6.xa0xa0 Mix the cell preparation and cryoprotective solution in equal portions. The final concentration will be 1.0 - 2.0 x 10⁷ cells/ml and 5% 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.

NOTE: To prevent culture contamination, penicillin-streptomycin solution (ATCC^® 30-2300x99) may be added to a final concentration of 50 to 100 I.U./ml penicillin and 50 to 100 µg/ml streptomycin.

7.xa0xa0 Dispense in 0.5 ml aliquots to 1.0-2.0 ml sterile plastic screw-capped cryovials.

8.xa0xa0 Place cryovials 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°xa0xa0xa0xa0xa0xa0 C/min.)

9.xa0xa0 Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator.

10.xa0xa0xa0xa0xa0xa0xa0xa0xa0 To thaw a frozen ampule, place it in a 35-37°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes. xa0Do not agitate the ampule.xa0 Do not leave ampule in water bath after thawed.

11.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of ATCC^® CCL-163x99 cells and 10 ml ATCC^® 30-2002x99 with 10% (v/v) HIFBS.

12.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Outgas the flask for 10 seconds with a 95% air, 5% CO₂ gas mixture.

13.xa0xa0xa0xa0xa0xa0xa0xa0xa0 Incubate in a 35-37°C CO₂ incubator with the cap screwed on tightly.