Parasite culture and growth inhibition assay

Malaria parasite P. falciparum strain 3D7 and chloroquine resistant strain RKL9 were cultured using O + packed erythrocytes using complete RPMI 1640 supplemented with AlbumaxII (Gibco, USA), hypoxanthine (Sigma-Aldrich, MA, USA) and gentamycin (Gibco, USA) in 37 °C incubator containing a mixed gas composition of 2% O₂, 5.5% CO₂ and 92.5% N₂. Parasites were synchronized using 5% Sorbitol (Sigma-Aldrich, USA) in two consecutive cycles. Assay was set up with tightly synchronized ring stage parasite culture with 1% parasitemia and 2% hematocrit in 96 well microtiter plates. Different concentrations of TMX (Cayman Chemical, USA) ranging from 2 µM to 20 µM (2 µM, 4 µM, 6 µM, 8 µM, 10 µM, 12 µM, 14 µM, 16 µM, 18 µM, 20 µM) were added to the parasites and the plates were incubated in 37 °C incubator containing a mixed gas composition mentioned earlier. Growth and invasion of the parasites were monitored from Giemsa (Sigma, USA) stained smears prepared at different time points. Invasion of the parasites were monitored using SYBR green dye assay at 48 h post invasion. Fluorescence was measured using microtiter plate reader (Varioskan Flash, Thermo, USA) with excitation at 485 nm and emission at 530 nm. Calculation of growth inhibition (% Inhibition) was done using the following formula: % Inhibition = [1 − % Fluorescence intensity (Treatment)/% Fluorescence intensity (Control)] × 100. IC₅₀ was determined by plotting the respective percent inhibitions of different concentrations of the drug using Graphpad Prism5 (CA, USA) software in non-linear regression mode.