Bright glow reagent

Human hepatic cells (12 × 10³; HepG2-A16-CD81-EGFP), stably transformed to express a GFP-CD81 fusion), were pretreated for 18 h with decreasing concentrations of the compounds of interest, over the range 50 µM to 0.85 nM. The cells were then infected with freshly dissected luciferase-expressing P. berghei (PbLuc) (4 × 10³) sporozoites^{51 (link)}. After 48 h of incubation with the compound, the viability of P. berghei exoerythrocytic forms (EEF) was measured by bioluminescence using Bright Glow reagent (Promega). HepG2 cytotoxicity was assessed by adding CellTiterGlo reagent (Promega). The plates were read in a PHERAstar FSX reader (BMG LABTECH).

Titration of pseudoviruses was carried out on TZM-bl cells, which contain the HIV-sensitive luciferase transgene, according to the protocol described by Sarzotti-Kelsoe [13 (link)]. One hundred microliters of cDMEM were added to each well of the 96-well plates. Then, 25 μL of the fivefold serial dilutions of pseudoviral stock were added. Then, a 100 μL suspension containing 10,000 TZM-bl cells in cDMEM with 8 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. All samples were assayed in triplicate. Twenty-four hours after transduction, the medium was changed to cDMEM. Seventy-two hours after transduction, the luciferase activity was estimated using a Bright-Glow reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The luminescence level was measured on a Tecan Infinite 200 Pro luminometer (Tecan Group Ltd., Männedorf, Switzerland). Then, an RLU (relative luminescence units) versus virus dilution curve was plotted, and the volume required to achieve a luminescence of about 200,000 RLU was determined. This volume was used in the following neutralization assays.