An adaptation of the
protocol by Linares et al. was undertaken.33 (link) Briefly, a P. falciparum 3D7 culture
was treated with the selected drug at 10 × EC50 using
the EC50 that was previously determined using the standard
72 h Pf asexual stage LDH assay. Treatment conditions were similar
to the ones used in the standard EC50 determinations (2%
hematocrit, 0.5% parasitemia). Three time points for drug treatment
were assessed, and samples of parasites were taken from the treated
culture at 0, 24, and 48 h time points. The drug was renewed every
24 h during the entire treatment period by taking out old media and
replenishing with new culture media with a fresh drug. After corresponding
drug treatment time, the drug was washed out, and drug-free parasites
were cultured by adding fresh erythrocytes pre-labelled with 10 μM
carboxylfluorescein diacetate succinimidyl ester (CFSE) and incubated
for a further 48 h. Upon completion of the 48 h, parasites were labelled
with 2 μM Hoechst and analyzed by flow cytometry (BD FACS Symphony)
with blue (515/20) + UV (450/50) laser. Newly invaded parasites were
gated by single cell erythrocytes and double positive for CFSE and
Hoechst staining (Q2). Viable parasites were calculated as follows:
viable parasites (%) = [Q2 (drug treated)/Q2(untreated)] × 100.
protocol by Linares et al. was undertaken.33 (link) Briefly, a P. falciparum 3D7 culture
was treated with the selected drug at 10 × EC50 using
the EC50 that was previously determined using the standard
72 h Pf asexual stage LDH assay. Treatment conditions were similar
to the ones used in the standard EC50 determinations (2%
hematocrit, 0.5% parasitemia). Three time points for drug treatment
were assessed, and samples of parasites were taken from the treated
culture at 0, 24, and 48 h time points. The drug was renewed every
24 h during the entire treatment period by taking out old media and
replenishing with new culture media with a fresh drug. After corresponding
drug treatment time, the drug was washed out, and drug-free parasites
were cultured by adding fresh erythrocytes pre-labelled with 10 μM
carboxylfluorescein diacetate succinimidyl ester (CFSE) and incubated
for a further 48 h. Upon completion of the 48 h, parasites were labelled
with 2 μM Hoechst and analyzed by flow cytometry (BD FACS Symphony)
with blue (515/20) + UV (450/50) laser. Newly invaded parasites were
gated by single cell erythrocytes and double positive for CFSE and
Hoechst staining (Q2). Viable parasites were calculated as follows:
viable parasites (%) = [Q2 (drug treated)/Q2(untreated)] × 100.
