Agilent Seahorse XFe96 Glycolysis Assay

An Agilent Seahorse XFe96 Analyzer was used to assess the metabolic output of adult and juvenile schistosomes in response to AMPK modulating factors. To determine the rate of glycolysis, the extracellular acidification rate was measured over time using the Agilent Seahorse XFe96 Extracellular Flux Assay Kit cartridge (Agilent Technologies, catalog no. 102905-100) and Agilent Seahorse XFe96 Spheroid Microplates (Agilent Technologies, catalog no. 102978-100). Twenty-four hours prior to the start of an experiment, cartridge probes were soaked in 200 μl Ultrapure Distilled Water (Invitrogen) at 37°C. The water was replaced with 200 μl Agilent Seahorse XF Calibrant (Agilent Technologies, catalog no. 100840-000) an hour prior to starting the experiment. Adult schistosomes were recovered by perfusion from infected mice immediately prior to the start of the experiment and washed with 1 × PBS to remove perfusion fluid. Either a single schistosome pair, containing one male and one female adult schistosome, or 500 newly transformed schistosomula were placed in each well of an Agilent Seahorse XFe96 Spheroid Microplate with 180 μl of Seahorse XF DMEM medium (Agilent Technologies, catalog no. 103575-100) supplemented with 1 mM Seahorse XF pyruvate solution (Agilent Technologies, catalog no. 103578-100), 10 mM Seahorse XF glucose solution (Agilent Technologies, catalog no. 103577-100), 2 mM Seahorse XF L-glutamine solution (Agilent Technologies, catalog no. 103579-100), and 1× antibiotic/antimycotic. Cartridge compound-administration chambers were loaded with 25 μl of either media or AMPK modulating compounds at pre-calculated concentrations to achieve desired final concentrations in a final volume of 205 μl of media before beginning Seahorse XFe96 Analyzer experiment.