NaCl: 4.2 g
Na3citrate•2H2O: 8.0 g
Glucose: 20.5 g
Glass distilled H2O to: 1.0 L
Dissolve components in glass distilled H2O, adjust the pH to 6.1 with 10% (w/v) citric acid and filter sterilize. The solution can be obtained from Sigma-Aldrich (cat# A3551).
Harvest and Preservation
Prepare a 30% (v/v) sterile glycerol solution in Alsever's solution.
Draw approximately 0.1 mL of anticoagulant solution (Yaeger's or heparin, etc.) into a syringe and move it back and forth over the length of the syringe, several times. Remove all air bubbles. Draw blood by cardiac puncture into the syringe from a host animal that has reached or is near peak parasitemia. If clotting occurs during extraction of blood, insufficient heparin was used.
Mix the heparinized blood with the 30% glycerol solution in a 2:1 ratio. If any clotting has occurred do not use. After mixing, the final concentration of cryoprotectant solution will be 10% (v/v). The mixture should be placed in a 4°C ice bath. The time from the mixing of the cell preparation and glycerol stock solution before the freezing process is begun should be no less than 15 min and no longer than 30 min.
Dispense in 0.5 mL aliquots into 1.0 mL to 2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation). Filled ampules should be placed in a 4°C ice bath. Do not immerse ampules to the level of the vial cap.
Plunge ampules from 4°C into liquid nitrogen. The frozen preparations may be stored in a mechanical freezer until needed, however storage in either the vapor or liquid phase of a nitrogen refrigerator is recommended for the longest viability.
To thaw a frozen ampule, place in a 35°C water bath, until thawed (2 to 3 min). Immerse the ampule just sufficient to cover the frozen material. Do not agitate the ampule.
- Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected mouse. Follow the protocol for maintenance in vivo. The course of infection may depend on the recovery of the parasite from the frozen state and the immune status of the host prior to infection.