Parasite growth inhibition assay

The effect of phosphatase inhibitors on parasite growth was evaluated on the 3D7 strains of P. falciparum. The parasite culture was synchronized using 5% sorbitol, and the assay was started at the schizont stage with hematocrit and parasitemia of synchronized schizont stage culture adjusted to 2% and 1%, respectively. Novartis_003209 (0.07–10 μm) was added to the parasite culture in separate 96‐well plates, and parasitemia was estimated after an incubation period of 24 h using flow cytometry. Briefly, cells from the samples were collected and washed with phosphate‐buffered saline (PBS), followed by staining with ethidium bromide (10 μg·mL⁻¹) for 20 min at 37 °C in dark. The cells were subsequently washed twice with PBS and analyzed on FACSCalibur (Becton Dickinson, USA), using cellquest software (Becton Dickinson, USA). Fluorescence signal (FL2) was detected with the 590 nm band‐pass filter using an excitation laser of 488 nm, collecting 100 000 cells per sample. Uninfected RBCs stained in a similar manner were used as a control. Following data acquisition, each sample was analyzed for percentage parasitemia by determining the proportion of FL2‐positive cells using CellQuest.