Figure 2

Reporter signal increases with both DNA and Na ₂ ATP concentration. (A) RBCs were transfected in a 96-well electroporation plate with 31 different pulses, in the three different buffers, SE, SF and SG. RLUC reporter activity in each well of the plate is shown as an intensity heatmap. (B) Set of pulses tested and their corresponding position on the electroporation plate. Position H4 is the pfGNr transfected negative control (pulse CM-150) (C) Increasing amounts of RLUC reporter plasmid DNA (pTI15) (2.5 μg, light gray diamond; 5 μg, dark gray square; 10 μg, black triangle) and increasing concentrations of Na₂ATP (0, 3, 6, 9, 12.5 mM) in SE buffer were mixed with 6 μl packed RBCs. Four independent transfection wells were assembled for each condition. Renilla luciferase activity was measured 48 h post-transfection. RLU, relative luminescence units. Error bars, s.e.m. (D) Transfection efficiency of single cuvette and plate-based methods. For the single cuvette transfection, the previously published protocol was followed [13]. The plate-based transfection was performed using the optimized protocol reported here (see Methods). Late stage parasites were added to pooled, single cuvette or 96-well, eRBCs and the mixture was re-split into four wells of a flat bottom 96-well culture plate eliminating outgrowth differences. Luciferase activity was measured in the four culture wells, normalizing for the number of parasites. Error bars, s.d.