Bivariate, high-content screening of Brugia malayi microfilariae

Abstract The following protocol is for performing bivariate (motility and viability) phenotypic drug screens against Brugia spp. microfilariae (mf). It has been heavily optimized for utilization with an ImageXpress high-content imager, but only small adaptations will be necessary for usage with similar imaging platforms.Mf are used immediately after receipt/extraction, and the experiment should not be run longer than 48 hours, after which mf health rapidly deteriorates. We have had success recording motility at 12 hours post-treatment (hpt) and again at 36 hpt, after which mf are immediately stained for viability, as well as 24/48 hpt time points.The two phenotypes could be assayed separately, but we have found that not every compound that causes reduction in mf motility causes a concomitant loss of viability. Thus, the discriminatory power of the pipeline is increased by running both assays in parallel, using the same plates. We have run the experiment using 16 plates at a time which will necessitate several hours of imaging at the desired time points. If several plates are planned, drug addition will need to be staggered every 5-6 minutes in accordance with the time taken to completely image each plate.Image data can be analyzed via a variety of software, but we have designed wrmXpress for this purpose (1).