In vitro P. berghei-Luciferase liver stage assays

Human hepatoma, HepG2-A16-CD81EGFP, cells were cultured at 37 °C in 5% CO₂ in DMEM media (DMEM (Life Technology, CA) supplemented with 10% FBS and 1x Pen-Strep-Glutamine (Gibco, CA). For both P. berghei-Luciferase and HepG2 cytotoxicity assays, 20–26 h prior to sporozoite infection, HepG2-A16-CD81EGFP20 cells (6 × 10⁵ cells/ml in assay medium; DMEM without Phenol Red (Life Technologies, CA), 5% FBS, and 5x Pen-Strep-Glutamine (Gibco, CA)) were seeded into white solid bottom 1536-well plates (custom GNF mold ref# 789173-F, Greiner Bio-One) at 5 μL per well. Compounds (50 nL in DMSO) were transferred to wells using an Acoustic Transfer System (Biosero). Atovaquone and Puromycin were used as positive controls for P. berghei-Luciferase liver stage assays and HepG2 cytotoxicity assays, respectively, and tested starting at a highest concentration of 10 μM, with 1:3 serial dilutions. DMSO (0.5% DMSO final concentration) was used as negative control for both assays. P. berghei-ANKA-GFP-Luc-SMCON (P. berghei-Luciferase)⁸⁸ sporozoites were freshly dissected from infected Anophelese stephensi mosquitoes received from the Insectary Core Facility at New York University (NYU). Sporozoites were then filtered twice through a 20 μm nylon net filter (Steriflip, Millipore) and 200 sporozoites/1 μL seeded into assay media. HepG2-A16-CD81EGFP cells were infected with 1 × 10³ sporozoites per well (5 μL) using a single tip Bottle Valve liquid handler (GNF). Plates were centrifuged at 24 °C for 3 min at 330×g on normal acceleration and brake settings (Eppendorf 5810 R centrifuge). HepG2-A16-CD81EGFP cells for toxicity assessment were left uninfected, with 5 µL assay media added to each well to match test plates. Plates were then incubated at 37 °C for 48 h in 5% CO₂ with high humidity chamber. After the incubation, P. berghei-Luciferase exoerythrocytic form growth and HepG2-A16-CD81EGFP cell viability were assessed by a bioluminescence measurement by spinning the inverted plates at 150xg for 30 s and adding 2 μL per well of Bright-Glo (Promega) for quantification of P. berghei-Luciferase exoerythrocytic forms or CellTiter-Glo (Promega) (diluted 1:2 with deionized water) for quantification of HepG2-A16-CD81EGFP cell viability. Immediately after addition of the luminescence reagent, the luminescence was measured by the Envision Multilabel Reader (PerkinElmer). For IC₅₀ determination, the background for the P. berghei-Luciferase growth inhibition was defined as average bioluminescence of the 16 wells with atovaquone (10⌠M), and the background for the HepG2 Cytotoxicity was determined as the average of the 12 wells with puromycin at single concentration (10⌠M). The baseline was defined as average bioluminescence of the 150 wells with 0.5% DMSO. IC₅₀ values were determined using the average normalised bioluminescence intensity of eight wells per concentration from two technical replicates of the 1536- well plates, and a nonlinear variable slope four-parameter regression curve fitting model in Prism 6 (GraphPad Software Inc.).