RPMI 1640 medium adjusted to contain 10% (v/v) heat-inactivated human serum (pooled Type A), 25 mM HEPES, 2 mM L-glutamine, 2 grams per liter D-glucose, 27 µg per mL hypoxanthine and 5 µg per mL gentamicin (optional)Human serum (pooled Type A or Type O recommended)
Please see Appendix II for complete medium preparation instructions and notes.
Incubation:
Temperature: 37°C
Atmosphere: 90% N2, 5% CO2, 5% O2
Propagation:
1. Place the frozen vial in a 37°C water bath until the culture is completely thawed. Transfer the vial to a biological safety hood and wipe the outside surface of the vial with 70% ethanol.
2. Using a sterile 1 mL pipette, aseptically transfer the contents of the vial to a sterile 50 mL conical centrifuge tube.
3. Add 12% sodium chloride (NaCl) solution dropwise, approximately 1:5 ratio NaCl to cell mixture (0.2× original culture volume). Allow to stand for 5 minutes.
4. Using a 1 mL syringe and 27-gauge needle, add dropwise while shaking 10 volumes of a 1.6% NaCl solution (10:1 ratio NaCl to original culture volume).
5. Centrifuge at 1000 × g for 5 minutes and remove most of the supernatant, leaving approximately 0.5 mL to 1 mL to resuspend the cell pellet. Resuspend the cells by gently swirling the tube.
6. Add dropwise while shaking 10 volumes of complete medium. Centrifuge at 1000 × g for 5 minutes and carefully remove the supernatant.
7. Add 5 mL of complete medium and transfer the sample to a 25 cm² tissue culture flask.
8. For continuous culture, add uninfected red blood cells (RBCs) to a 1% to 2% hematocrit solution (immediately or the next day).
9. Gently aerate culture with a 90% N2, 5% CO2, 5% O2 gas mixture through a sterile, cotton-plugged Pasteur pipet. Incubate the flask at 37°C.
10. Take a smear for Giemsa staining after 1 day to evaluate parasite growth and determine parasitemia.
Maintenance:
Note: Changing of the culture medium every 1 day is required for malaria-infected erythrocyte cultures.
1. Remove the flask with infected culture from the 37°C incubator and place onto a flask warmer.
2. Carefully remove the supernatant with a sterile, unplugged Pasteur pipet under vacuum. Remove as much of the supernatant as possible without taking the cells.
3. Add 25 mL of sterile warm (37°C) complete medium to the flask, gently mix and aerate, then quickly tighten the cap and place the flask in the 37°C incubator until the next change of medium.
Preparation of Blood Smear:
1. Carefully remove 0.5 mL to 1 mL of mixed culture with a sterile pipet and transfer to a microcentrifuge tube.
2. Centrifuge the microcentrifuge tube at high speed and aspirate the supernatant.
3. Mix the pellet and transfer 6 µL of the suspension to a glass slide for a thick film smear or 2 µL for a thin film smear. Spread the drop into a thin film using the edge of a clean glass slide. Air dry for 3 minutes at room temperature.
4. Fix the blood smear by rinsing it with methyl alcohol. Air dry for 3 minutes at room temperature.
5. Stain blood films in 10% Giemsa solution for 15 minutes. Rinse with distilled water and allow to air dry.
6. Using light microscopy at 100× magnification, determine parasitemia of culture.