High-Content Imaging of Hepatocytic P. falciparum

Intrahepatocytic development of P. falciparum NF54 parasites was studied by seeding primary human hepatocytes at 60,000 cells/well and culturing for 2 days in collagen-coated 96-well plates. They were then overlaid with 50,000 P. falciparum NF54 sporozoites and compounds. The supernatant was refreshed daily with fresh compounds. Four days postinfection, parasites were visualized by fluorescent (Alexa Fluor 546, Invitrogen) immunostaining of the Hsp70 antigen, and host and parasite nuclei were visualized by Hoechst 33342 DNA staining. Parasites and host cells were quantified and sized using an ImageXpress Pico high-content microscope using Cell Report Xpress software. Object parameters for quantification of intensity and area were calculated automatically based on user-selected cells of interest and were adapted, where necessary, to achieve a clear distinction between positive (100 nM atovaquone) and negative (0.1% DMSO) control wells. Recognition of parasites was based on the overlap of Hoechst and Alexa Fluor 546 signals. Using parasite strain NF175²⁵ , the assay was miniaturized to a 384-well plate format. Procedures were similar except that 18,000 primary hepatocytes were seeded per well and, following culturing for 2 days, overlaid with 10,000 sporozoites.