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MicroPubl Biol,
2020]
The purpose of this study was to determine the utility of the wMicroTracker as a screening platform to assess the motility of various parasites. We tested three species of parasites: the adult and larval stages of the filarial nematode Brugia pahangi, the schistosomula stage of the trematode Schistosoma mansoni, and the epimastigote stage of the protozoan parasite Trypanosoma cruzi. We optimized the assay for the number of parasites per well, plate type and media volume using the wMicroTracker and compared those readouts to readouts from the WormAssay (Marcellino et al. 2012) when possible. The WormAssay has been used in phenotypic drug screens to identify new compounds for the treatment of lymphatic filariasis, onchocerciasis and schistosomiasis (Storey et al. 2014; Bulman et al. 2015; Weeks et al. 2018; Tyagi et al. 2019). The original WormAssay was developed by Marcellino et al. 2012 and was subsequently modified to the Worminator by Storey et al. 2014 to observe smaller worms with an inverted microscope. wMicroTracker (InVivo Biosystems) protocols optimized for C. elegans adults (1 mm in length by 80 m in width, highly motile) were used to optimize parasite assays based on the size and motility of each of the parasite species as compared to C. elegans adults.To use the wMicroTracker, two factors need to be considered: the size of the parasite of interest and how active they are. The wMicroTracker detects movement when an organism crosses the stationary LED beam at the center of the well. Hence, parasites that do not travel throughout the well should be assayed in a U-bottom plate to ensure movement is detected. For parasites that do travel throughout the well, a flat bottom plate is sufficient. To determine the number of parasites to use per well, we compared their size to that of C. elegans adults. Following the InVivo Biosystems protocol (https://invivobiosystems.com/wp-content/uploads/2018/12/Protocol_toxicity-in-c-elegans_122018.pdf), C. elegans adults were screened in the wMicroTracker with 40-50 adults per well in 100 L of M9 buffer (Stiernagle 2006). This produced results in the range of 25-35 mean movement units per well (Figure 1A). To optimize screening methods in the wMicroTracker for other parasites, the size and motility of parasites of interest were compared to C. elegans adults to ensure control wells gave mean movement units around this range.B. pahangi females (34.7 mm in length by 139 m in width (Mutafchiev et al. 2014), highly motile) were screened with one worm per well in 500 L of RPMI in a 24-well flat bottom plate with 8-9 replicate wells. B. pahangi males (18.0 mm in length by 77 m in width (Mutafchiev et al. 2014), high motility) were screened with four worms per well in 500 L of RPMI in a 24-well flat bottom plate with 4-7 replicate wells. B. pahangi females and males were assessed in both screening platforms. Both platforms showed similar motility profiles in response to 50 M ivermectin or 1% DMSO as a negative control (Figure 1B