Bp0352

To demonstrate the ability of L3 and L4 A. suum to ingest fluorescent probes, two function-based fluorescent probes were used in feeding assays: 1) beta-ala,lys-AMCA (BAL-AMCA), 200 μM (BP0352, BioTrend, Zurich, Switzerland) (Meissner et al., 2004 (link)); and 2) DQ Green-BSA (D-12050, Molecular Probes, Eugene OR), 100 μg/mL (Vazquez and Colombo, 2009 (link)). L3 and L4 were cultured in the presence of these probes for 4 h prior to assessment of ingestion. Bisbenzimide (BB,; Hoechst nuclear dye 33258, Sigma-Aldrich, St. Louis MO) was used at 10 μg/mL and incubated with larvae for 4 h prior to assessment by fluorescent microscopy to document whole larva inventories of nuclei in cells and organs observable with this dye. Propidium iodide (PI, P4170, Sigma-Aldrich, St. Louis MO) was used at 100 μM, and incubated along with BB for 4 h prior to addition of experimental treatments. Preincubation was intended to maximize access of tissues to the fluorescent probes in presence of various treatments. For most experiments, larvae were then incubated with the two (BB and/or PI) fluorescent probes and the treatment for the duration of the experiment.

To determine the ability of xL3 and L4 H. contortus to ingest fluorescent probes, two function-based fluorescent probes were used in feeding assays: (1) beta-ala,lys-AMCA (BAL-AMCA), 200 μM (BP0352, BioTrend, Zurich, Switzerland) [12 (link)]; and (2) DQ Green-BSA (D-12050, Molecular Probes, Eugene, OR, USA), 100 μg/mL [13 (link)]. L3 and L4 were cultured in the presence of these probes for 4 h prior to assessment of ingestion. To assess staining of nuclei by bisbenzimide (BB, 10 µg/mL, H 33258, Sigma-Aldrich, St. Louis, MO, USA) live xL3 and L4 were incubated with BB in standard culture conditions for 16 h prior to assessment by fluorescent microscopy. To assess cell death in whole xL3 and L4, propidium iodide (PI, P4170, Sigma-Aldrich, St. Louis, MO, USA) was used at 100 μM, and added to larval cultures 4 h prior to the addition of experimental treatments. Preincubation leading to ingestion of PI was intended to maximize access of tissues to the fluorescent probes in presence of various treatments.