Centricon plus 70 filter

For purification of recombinant Nrx1α wild-type or ΔHS proteins, pcDNA4-Nrx1α-PLAP-Myc-His or pcDNA4-Nrx1αΔHS-PLAP-Myc-His were transfected into HEK293 cells which were cultured in DMEM with 10% FBS and selected for four weeks in the presence of 0.5 mg/ml Zeocin. The resulting Zeocin-resistant cells were grown in a serum-free AIM V synthetic medium for four weeks during which medium was collected every three days.
For the rest of the recombinant proteins in this study, the protein expression constructs were transfected into HEK293 using PEI “Max” (Polysciences, Inc.). After 24 hours post-transfection, the HEK293 cells were cultured in a serum free DMEM medium for 48 hours.
The harvested condition medium was first concentrated in PBS with Centricon Plus-70 filters (Millipore). The recombinant His tag proteins were purified by binding to Ni-NTA agarose beads (QIAGEN) and eluted with 200 mM imidazole. The recombinant Fc tag proteins were purified by binding to protein G beads (GE Healthcare) and eluted with 0.1 M glycine (pH 2.5) followed by a rapid addition of 1/10 volume of TrisHCl (pH 8.6). The purified proteins were washed with PBS and concentrated using Amicon Ultra-15 centrifugal filter units (Millipore) with the appropriate protein cut-off size. Recombinant protein concentrations were quantitated by SDS-PAGE with bovine serum albumin standards using Sypro Ruby gel stain.

EPC-secretome obtained by LSP (HD-CM and SP-CM) or HSP (CM), and fresh protein-free EBM (control) were filtered through a 0.22-μm vacuum filter to remove cells and debris. Centricon Plus-70 filters (3 kDa Ultracel-PL membrane—Millipore) were filled with EPC-secretome or EBM and centrifuged (3500×g) at 4 °C for 60 min. The super-concentrated EPC-secretomes (scHD-CM, scSP-CM, scCM) or EBM (scEBM) was recovered by centrifuging in collection mode at 1000 g for 1 min. Finally, the protein content was determined by Bradford assay and frozen at − 80 °C in low-protein binding tubes.

Three independent experiments were done with yeast and hyphal cultures. The isolation of EVs was done according to Gil-Bona et al. (21 (link)). The whole process was conducted at 4°C. In brief, the supernatants from 1-L volumes of yeast- and hypha-specific culture media grown during 16 h at 37°C and 180 rpm were collected by 20 min of centrifugation at 8,000 rpm at 4°C in a Beckman Coulter J2-HS centrifuge using the JA-10 rotor. The supernatants were then filtrated using a 0.45-μm filter to ensure the elimination of all the cells and cell debris. One protease inhibitor tablet (Pierce, EDTA-free; Thermo Fisher) along with 1 mL of phenylmethanesulfonylfluoride (PMSF) was added to each of the 1-L volumes of filtrated supernatants. These supernatants were concentrated afterwards, using a Centricon plus-70 filter (100-kDa-cutoff filter; Millipore), by centrifugation at 2,500 rpm in an Eppendorf 5810R centrifuge to a final volume of 8 mL. The concentrated supernatants were subsequently ultracentrifuged at 100,000 × g (34,200 rpm) for 1 h at 4°C in a Beckman Optima XL-90 using a 90 Ti rotor. The pellets containing the isolated EVs were washed twice with PBS and solubilized in 50 μL of 0.5 M triethylammonium bicarbonate (TEAB) buffer. Protein concentration was measured using the Bradford protein assay (Bio-Rad), following the manufacturer’s instructions.

Infectious HBV particles were collected from HepDE19 cells (a gift from Haitao Guo, Indiana University) as previously described (Michailidis et al., 2017 (link)). Briefly, HepDE19 cells were cultured in tetracycline-free medium to induce HBV virion production. Supernatant was collected every other day and replenished for 20 days. The collected supernatant was concentrated with Centricon^® Plus-70 Filter (Millipore, United States) by centrifuging with 2,500 × g for 30 min at 4°C. The concentrated HBV particle stocks were stored at −80°C. HBV inoculum was determined by quantitative PCR (qPCR) of HBV DNA. The quantification of HBV inoculum was expressed as HBV genome equivalent/cell (geq/cell), which was equal to HBV DNA copies. The protocol for HBV DNA quantitation was described before (Shlomai et al., 2014 (link); Xiang et al., 2017 (link)).