Determination of IC50

Determination of IC50

Posted on Saturday, January 17, 2004

Description
In vitro whole cell assay by [H3] Hypoxanthine uptake assay

Procedure
[H3] Hypoxanthine uptake assay (with respect to red blood cell cultures of P. falciparum
Dispense 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 wells
Add 200 ul of parasitized RBC to all wells (1.7% hematocrit)
Incubate at 37C for 24 hours
Add 25 ul [H3] hypoxanthine (0.5mCi) solution to each well
Incubate for next 18 hours
Harvest the plate using MASH type harvester
Allow filter strips to dry, cut out filter disks and place the disks into scintillation vials (with scintillation fluid)
Count activity in a liquid scintillation counter
The 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)