Orthogonal ex vivo Brugia validation assay

Adult B. pahangi and B. malayi females cultivated in and extracted from peritoneal cavities of jirds (Meriones unguiculatus) were obtained mainly from TRS Laboratories. B. pahangi were also provided by B. T. Beerntsen (University of Missouri) and the National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (NIAID) Filariasis Research Reagent Resource Center for distribution by BEI Resources, NIAID, NIH [adult female B. pahangi (live), NR-48903]. After shipment, worms were immediately separated into 24-well plates, one animal per well, and allowed to recover for 2 days in high-glucose RPMI 1640 medium (the American Type Culture Collection modification; Gibco) supplemented with 10% minimum essential medium (Gibco) and 10% heat-inactivated HyClone FBS (GE Healthcare Life Sciences). Media were changed daily, and compounds were tested at indicated concentrations (0.1% DMSO). Gross motility of worms was observed by eye during treatment and compared to DMSO controls. After 3 days of treatment, animals were frozen at −80°C, thawed, and fixed for 20 min with 3.2% PFA in PBS-T. Ovaries were dissected out, stained for Wolbachia using a modified FISH protocol, mounted on slides using VECTASHIELD with DAPI mounting medium (H-1200, Vector Laboratories Inc.) and imaged using a confocal microscope (see Supplementary Materials and Methods). To reduce variability, worms originating from a single jird were used in each experiment. The experiments were carried out partially blinded because, with the exception of DMSO and doxycycline controls, the identity of tested compounds was masked during treatment, imaging, and analysis.