Amicon ultra centrifugation filter

The principle of the MAF adsorption/elution method was based on the procedure as previously described [18 ]. Monolithic discs, diameter 3.86 cm and height 1.0 cm, were synthesized by polymerization of polyglycerol-3-glycidyl ether (Ipox chemicals, Laupheim, Germany). An 80:20 mixture of toluene and tert-buthyl methyl ether was used as porogen to create monoliths with a pore size of ca. 20 μm. After synthesis, functionalization was performed by recirculating 10% diethylamine in 50% ethanol at 60°C through the monolithic disks for 3 h to create positively charged diethylaminoethyl groups on the pore surface. Afterwards the monoliths were rinsed with ultrapure water and stored at 4°C until further use. One liter of raw sewage was filtrated through a MAF disc (Microarray and Bioseparation Group of the Institute of Hydrochemistry, Technical University of Munich, Germany) assembled as previously described [28 ]. Viruses were eluted from the filter by soaking 2×2 min in a total of 20 mL high salt buffer (1.5 M NaCl, 0.05 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 7). The eluate was further concentrated to 3 mL by 100 kDa Amicon ultra centrifugation filters (Merck Millipore, Cork, Ireland) according to the manufacturer’s instructions. The viral concentrate was stored at -80°C until further processing.

Acanthamoeba castellanii was cultured in PYG medium in T-75 flasks until they reached 80% confluency, followed by removal of the culture medium. The cells were washed three times and resuspended in Pageʼs modified Neffʼs amoeba saline (PAS) (120 mg NaCl, 4 mg MgSO₄ × 7H₂O, 3 mg CaCl₂, 142 mg Na₂HPO₄, 136 mg KH₂PO₄ in 1 l distilled water) for 48 h. The cell debris was removed by centrifugation at 2000×g for 30 min and further centrifuged at 10,000×g for 30 min. The cell-free medium was ultra-filtered through Amicon ultracentrifugation filters (Merck Millipore, Billerica, MA, USA), then filtered through a 0.22 μm filter. Next, 0.5 volumes of the total exosome isolation reagent (Life Technologies, Carlsbad, CA, USA) were added to the supernatants and incubated at 4 °C overnight. After incubation, the supernatants were centrifuged at 10,000×g for 1 h. Pellets were resuspended in 50 μl PBS and stored at − 80 °C. The concentration of exosome protein was determined by the Bradford assay (Bio-Rad Laboratories, Inc., USA). The protein samples were separated with 10% SDS-PAGE (T-Pro, Taipei, Taiwan) followed by silver staining using standard procedures.

Poly(3-hydroxybutyric acid-co-hydroxyvaleric acid) (PHBV) 12% w/w poly-3-hydroxyvalerate (PHV); polyvinyl alcohol (PVA) (average mol wt. 30,000–70,000); Escherichia coli O55 lipopolysaccharide: B5; TWEEN 20 and 0.45 µm Millipore Filters were purchased from Sigma-Aldrich (St. Louis, MO). Trypan Blue stain 0.4%; antibiotic–antifungal (100×); bovine fetal serum; trypsin-EDTA 1× and Nile Red 552/636 were purchased from Gibco by Life Technologies (Carlsbad, CA). Hoechst 33342 was purchased from Invitrogen (Carlsbad, CA). EDTA was purchased from Calbiochem (San Diego, CA). PBS was purchased from Winkler (Taipei City, Taiwan). Dichloromethane, sodium bicarbonate, sulfuric acid, hydrogen peroxide, Triton X-100, and Amicon Ultra centrifugation filters were purchased from Merck (v). CytoTox 96 Non-Radioactive LDH Cytotoxicity Assay was purchased from Promega (Madison, WI). IL-6 Human ELISA MAX Deluxe Set, Biolegend (San Diego, CA); TNF-α Human ELISA MAX Deluxe Set, Biolegend, and Nunc MaxiSorp ELISA Plate were purchased from Biolegend (San Diego, CA). NOD1 Agonist γ-d-Glu-mDAP (iE-DAP) were purchased from Invivogen (San Diego, CA).

For the fractionation, the extracts were passed through a fast performance liquid chromatography column (Sephadex G-50, 1.0 × 48 cm) coupled to an ÄKTA purifier system (UPC 100, GE Healthcare). The samples (500 μg of protein/500 μL) were eluted with 1.6 mM acetic acid at a flow rate of 1 mL/min. The absorbance was read at 280 nm and fractions (10 mL) were collected and pooled according to the peaks observed in the chromatogram. Then, the peaks retained in the column were filtrated with Amicon® Ultra centrifugation filters with 10 K cutoff (Merck Millipore, USA). Spin filtration was executed as recommended by the manufacturer manual supplied with the product. The chosen centrifugation time was 20 min at 14,000 × g.

The molecular weight of the extracted fucoidan was estimated using analytical ultracentrifugation. A volume of 15 mL of E. radiata fucoidan stock solution (0.5 mg/mL) prepared in distilled water was transferred into and filtered through 100 K, 50 K, 30 K and 10 K Amicon^® ultra-centrifugation filters (MERCK). The retentates were obtained by centrifuging the filters at 4000 g for 20 min. The filtrate and retentate samples from each filtration step were analysed for the presence of fucoidan according to the phenol-sulfuric acid assay.