Inoculation:4
1. Thaw a frozen cryovial of MRA-680 in a 35°C to 37°C water bath for approximately 2 to 3 minutes. Do not allow the vial to immerse near the cap line seal while thawing.
2. Once thawed, wipe the outside of the vial with 70% ethanol before opening. Using a 1 mL syringe equipped with a 27-gauge 1/2 inch needle, remove approximately 200 µL to 300 µL from the vial.
3. Wipe the injection site of the mouse with 70% ethanol and inject the sample intraperitoneally at 50 µL to 100 µL per mouse (approximately 3 mice for most applications).
Monitoring parasitemia:
1. Starting 3 days post-inoculation, monitor the growth of parasites by tail vein bleed sampling and Giemsa-stained thin blood smear microscopy at 1- to 2-day intervals.
2. Passage the strain when the infection is at or near the first peak of parasitemia (> 5%). This will normally occur within one week of inoculation.
% parasitemia = (Infected RBC/Total RBC) × 100
Passaging:
1. Anesthetize infected mice by CO2/O2 inhalation. Collect the blood by orbital bleeding or from the tail vein into 25 mL of cold 1× PBS-heparin anticoagulant solution (please refer to Appendix I for preparation instructions).
2. Inject the sample into each of the uninfected mice (approximately 10 mice) as described in Inoculation step #3 above.
3. Monitor parasitemia as described above and passage as needed.
Please refer to Appendix I for cryopreservation instructions.
Note: Do not directly inject freshly thawed parasites from cryopreserved stocks by the intravenous (IV) route, as these samples contain cryoprotectant, anticoagulant and may contain traces of lysed or coagulated red blood cells. Direct IV inoculation from cryopreserved stock may result in pulmonary embolism or shock in mice.