Rapidvap vacuum dry evaporation system

Dry samples of dinospores (1 × 10⁶ dinospores per sample), tomonts (4 × 10⁵ tomonts per sample) and gills infested with trophonts (7.2 × 10⁶ trophonts per sample) were extracted with 3 mL of methanol (ACS reagent, ≥99.8%, Sigma^®, St. Louis, MO, USA) by sonication for 3 min at 70% amplitude and 3 s pulses (Vibra-Cell VCX 500, Sonics & Materials Inc., Newtown, CT, USA) while cooling on an ice bath. Cell disruption was monitored under a microscope; when 90 ± 1% of the cells were disrupted the extracts were centrifuged at 1700× g for 10 min. The supernatants were collected and fractioned by Solid Phase Extraction (SPE) cartridges (Supelclean LC-18 SPE cartridge, 3 mL, Supelco, Sigma-Aldrich^®, St. Louis, MO, USA), following the protocol previously described by Moeller et al., with modifications [27 ]. Briefly, cartridges were conditioned with 1 mL of 100% methanol and then, samples were loaded and fractionated using solvents of increasing polarity, where Fraction 1 corresponds to 100% methanol, Fraction 2 to 80% methanol, Fraction 3 to 50% methanol, Fraction 4 to 25% methanol and Fraction 5 to 0% methanol (100% water). The solvent was evaporated under vacuum at 60 °C before the hemolytic assays (RapidVap Vacuum Dry Evaporation system, Labconco, Kansas City, MO, USA).