To begin, obtain the sorbitol synchronized ring stage parasites of plasmodium falciparum. Prepare 50 milliliters of 2x cytomix buffer and dilute it 1 to 1 with sterile water. Then, wash 100 microliters of packed ring stage parasitized RBCs twice with 5 milliliters of 1X cytomix buffer and centrifuge them at 994G for three minutes at room temperature.
After removing the buffer, add 50 micrograms of each plasmid into the cells, followed by an appropriate volume of 2x buffer. Adjust the volume to 400 microliters with 1x buffer before transferring the prepared solution into 0.2 centimeter cuvettes for each transfection. Then, electroporate the cells carefully.
Following electroporation, mix and incubate the cells with complete RPMI medium in a T25 flask with a total volume of 15 milliliters. Next, centrifuge the transfected parasites in a 50 milliliter conical tube at 994G for three minutes. After removing the supernatant, wash the parasites with 10 milliliters of incomplete RPMI.
Culture them with fresh complete RPMI for two days, replenishing the medium after 24 hours. After 48 hours, remove the complete RPMI from each flask. Resuspend the culture in complete RPMI added with drugs.
Incubate the culture in a T75 flask along with 400 microliters of fresh red blood cells for a fortnight On day 14, aliquot 200 microliters of the transfected culture into a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 500G for five minutes at room temperature. After aspirating the supernatant, transfer the cells onto a slide and place another slide on it at a 45 degree angle to make a smear.
Following methanol fixation, immerse the slide in Giemsa stain for 20 minutes. Wash the slide under running water and dry it thoroughly. Observe the slide under a light microscope at 100x magnification after applying immersion oil.
Once the parasites are visualized, replenish the medium daily for two to three days.
