NF54-cg6-ULG8-CBG99 cultures induced to
produce gametocytes were treated with NAG for 96h, after which gametocytes were purified
from uninfected erythrocytes on MACS Separation Columns CS (Miltenyi Biotec) and allowed
to mature to stage V over the following 8 days. To calculate IC50 values,
compounds (Table 1 ) were serially diluted across
ten twofold dilutions and dispensed in 96-well plates in a final volume of
100μl/well. Synchronous 8×104 stage V gametocytes were
resuspended in 100μl of complete medium and incubated with the compounds at
37°C for the time indicated. To calculate the IC50 of MB in the
presence of a fixed dose of different compounds, MB was dispensed in 96-well plates as
described above. Gametocytes were dispensed as described above with 1 μM of the
different compounds and incubated with MB at 37°C. Cell viability was evaluated by
adding a non lysing formulation of 0.5 mM D-Luciferin substrate (Cevenini et al., 2014 (link)) and measuring luciferase
activity for 1 second on a Varioskan™ Flash Multimode Reader (Thermo Scientific).
The percent viability was calculated as a function of drug concentration. Curve fitting
was obtained by non-linear regression analysis (GraphPad Prism 6.0).
produce gametocytes were treated with NAG for 96h, after which gametocytes were purified
from uninfected erythrocytes on MACS Separation Columns CS (Miltenyi Biotec) and allowed
to mature to stage V over the following 8 days. To calculate IC50 values,
compounds (
ten twofold dilutions and dispensed in 96-well plates in a final volume of
100μl/well. Synchronous 8×104 stage V gametocytes were
resuspended in 100μl of complete medium and incubated with the compounds at
37°C for the time indicated. To calculate the IC50 of MB in the
presence of a fixed dose of different compounds, MB was dispensed in 96-well plates as
described above. Gametocytes were dispensed as described above with 1 μM of the
different compounds and incubated with MB at 37°C. Cell viability was evaluated by
adding a non lysing formulation of 0.5 mM D-Luciferin substrate (Cevenini et al., 2014 (link)) and measuring luciferase
activity for 1 second on a Varioskan™ Flash Multimode Reader (Thermo Scientific).
The percent viability was calculated as a function of drug concentration. Curve fitting
was obtained by non-linear regression analysis (GraphPad Prism 6.0).
