Determination of IC50

Determination of IC50

Posted on Saturday, January 17, 2004 DescriptionIn vitro whole cell assay by [H3] Hypoxanthine uptake assay Procedure[H3] Hypoxanthine uptake assay (with respect to red blood cell cultures of P. falciparumDispense 25 ul complete RPMI in different wells (triplicate for each drug concentration)Dispense 25 ul of drug with unknown antimalarial activity concentration range 1-10 uM in these wellsAdd 200 ul of parasitized RBC to all wells (1.7% hematocrit) Incubate at 37C for 24 hoursAdd 25 ul [H3] hypoxanthine (0.5mCi) solution to each wellIncubate for next 18 hoursHarvest the plate using MASH type harvesterAllow filter strips to dry, cut out filter disks and place the disks into scintillation vials (with scintillation fluid)Count activity in a liquid scintillation counterThe concentration response data are fitted to a sigmoid function using computerized non linear regression analysis (Rodhard et al, 1980) (Control wells with no drug as well as non parasitized RBC are also taken in the same microtiter plate)