Vivaspin 6 5000 mwco tubes

To obtain HES products, CBF1 mice were infected with L3 stage larvae by oral gavage, with adult parasites collected from the small intestine 14 days post-infection, as described previously (Johnston et al., 2015 (link)). Following isolation, adult H. polygyrus worms were kept in serum-free media in vitro, and the secretion product was collected every 3 days for 14 days. Eggs were removed by spinning at 400 × g before filtering through a 0.2-μm filter (Millipore). Purified medium was then spun at 100,000 × g for 2 hr in polyallomer tubes at 4°C in a SW40 rotor (Beckman Coulter). The ultracentrifuged pellet was washed twice in filtered PBS at 100,000 × g for 2 hr, and the final pellet was resuspended in PBS and protein content was quantified by Qubit (Invitrogen) and stored at −80°C. Qualitative analysis of EV purity was carried out using silver stain analysis as described previously (Buck et al., 2014 (link)). The resulting supernatant (and total HES product) was concentrated using Vivaspin 6 5000 MWCO tubes (Fisher) at 5,000 × g, and it was washed twice with PBS.

For collection of H. polygyrus secretion product, CF1 mice are infected with infective-stage larvae by gavage and adult parasites collected from the small intestine 14 days post infection. The worms are maintained in serum-free media in vitro as described elsewhere37 (link); secretion product is collected every 3 days for a maximum of 3 weeks (samples used here were from the first week of collection) and purified as follows: eggs are removed by spinning at 400 g before filtering of the secretion through 0.2-μm filter (Millipore). Filtered media is then processed following a modified protocol from that described in ref. 38 , by being spun at 100,000 g for 2 h in polyallomer tubes at 4 °C in a SW40 rotor (Beckman Coulter). Pelleted material is washed two times in filtered PBS at 100,000 g for 2 h. The supernatant is concentrated using Vivaspin 6 5000 MWCO tubes (Fisher) at 5,000 g and washed two times with PBS.