P. falciparum was propagated in
RPMI-1640 containing 0.5% albumax I as previously described.20 (link),22 (link) For EC50 determination, parasites (0.19 mL of 0.5% parasitemia,
0.5% HCT) were plated into 96-well microtiter plates containing 10
μL compound or DMSO control. The last column of each plate was
reserved for non-parasitized RBCs (0.5% HCT) to determine background
fluorescence. Serial dilutions of compound stocks were prepared in
100% DMSO at 200× the final concentration. After 72 h of incubation,
parasitized RBCs were quantitated by the SYBR Green method. 2×
SYBR Green I solution (20 μL) in 1× PBS was mixed with
20 μL of parasites in 96-well plates and incubated for 20 min,
after which time 160 μL of 1× PBS was added. Fluorescence
was detected using a BD Biosciences Acurri C6 flow cytometer, and
events were recorded within gates that encompassed all asexual growth
stages of the P. falciparum intraerythrocytic
life cycle. A minimum 50 000 total events were recorded per
well. Background events determined from non-parasitized RBC controls
were subtracted from final counts. All data were collected in triplicate.
RPMI-1640 containing 0.5% albumax I as previously described.20 (link),22 (link) For EC50 determination, parasites (0.19 mL of 0.5% parasitemia,
0.5% HCT) were plated into 96-well microtiter plates containing 10
μL compound or DMSO control. The last column of each plate was
reserved for non-parasitized RBCs (0.5% HCT) to determine background
fluorescence. Serial dilutions of compound stocks were prepared in
100% DMSO at 200× the final concentration. After 72 h of incubation,
parasitized RBCs were quantitated by the SYBR Green method. 2×
SYBR Green I solution (20 μL) in 1× PBS was mixed with
20 μL of parasites in 96-well plates and incubated for 20 min,
after which time 160 μL of 1× PBS was added. Fluorescence
was detected using a BD Biosciences Acurri C6 flow cytometer, and
events were recorded within gates that encompassed all asexual growth
stages of the P. falciparum intraerythrocytic
life cycle. A minimum 50 000 total events were recorded per
well. Background events determined from non-parasitized RBC controls
were subtracted from final counts. All data were collected in triplicate.
