Nonidet p 40

Total protein was extracted from microvesicles and exosomes in ice-cold buffer [50 mM TRIS (Sigma-Aldrich, MO, USA), 150 mM NaCl (Labsynth, SP, Brazil), 1.0% nonidet-P-40 (Bio-Rad Laboratories, CA, USA), 0.5% sodium deoxycholate (Sigma-Aldrich), and 0.1% SDS (pH 8.0; Sigma-Aldrich) containing protease inhibitors (AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A; Protease Inhibitor Cocktail; Sigma-Aldrich)] and quantified using a modified Lowry method (Bio-Rad, HH, UK). The protein samples (50 μg) were separated according to size by 12% SDS-PAGE and electroblotted onto nitrocellulose membranes (GE Life Sciences, LC, UK). The membrane blots were probed with primary antibodies overnight at 4°C and with HRP-conjugated secondary antibodies for 1 h at 4°C. The primary antibodies used were as follows: anti-MMP2 diluted at 1 : 100 (Abcam, MA, USA) for the MVs and anti-CD63 diluted at 1 : 100 (Abcam, CBG, UK) for the EXs. Next, the membranes were incubated with HRP-conjugated secondary antibodies (GE Life Sciences). The protein bands were visualized using the Immobilon Western HRP substrate (Millipore, MO, USA). The obtained bands were quantified using Uvitec analysis software (Uvitec Limited, CBG, UK).

F. hepatica tegumental extract (FhTeg) was prepared as previously reported (13 ). In brief, F. hepatica adult worms following collection from sheep at an abattoir in Ireland were washed in sterile phosphate-buffered saline (PBS) and incubated in 1% Nonidet P-40 (Nonidet P-40 (Sigma Aldrich, Wicklow, Ireland) in PBS) for 30 min. Supernatant was collected and Nonidet P-40 removed using detergent-removing biobeads (Bio-Rad laboratories, Fannin Ltd, Dublin, Ireland), and the remaining supernatant was centrifuged at 14,000 × g for 30 min at 4 °C prior to being filtered/concentrated using compressed air, and then stored at −20 °C. All protein concentrations were determined using a bicinchoninic acid protein assay kit (Promeaga, Madison, WI).