Optima xe 90 ultracentrifuge

OF-sEVs were obtained from the isthmus of ipsilateral oviducts from pregnant and non-pregnant groups according to the isolation protocol previously used by da Silveira et al. (53 (link)), Alminaña et al. (32 (link)), and Ávila et al. (54 (link)). Briefly, the oviductal flushing was centrifuged at 300 × g for 10 min to remove live cells, 2,000 × g for 10 min to remove cellular debris, 16,500 × g for 30 min to remove large vesicles, and the remaining supernatant was placed at −80°C until further use. The supernatant was filtered through a 0.20 μm sterile syringe filter (PES membrane; Corning) in order to remove any remaining large EVs. For sEVs isolation, filtered oviductal flushing was centrifuged twice at 119,700 × g for 70 min at 4°C (Optima XE-90 Ultracentrifuge; rotor 70 Ti; Beckman Coulter). After this procedure, the supernatant was discarded, and the sEVs pellets were resuspended in 20 μL of phosphate-buffered saline (1 × Ca²⁺/Mg²⁺ free PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) and used for further analysis. Due to the small volume of oviductal flushing obtained from a single animal, the protocol to isolate OF-sEVs was validated using female reproductive tracts obtained from slaughterhouses, as described in Supplementary Data 1.

Halobacterium salinarum S9 was grown as previously described, with no additional antibiotics necessary due to the culture conditions being too harsh for any contamination^{34 (link)}. The expression culture was grown at 38 °C with illumination in either an Erlenmeyer flask or a Winpact Bench-Top Fermenter (Major Science, Taiwan). The PM was purified from the bacterial pellet using a standard sucrose gradient procedure, in an Optima XE-90 Ultracentrifuge with the SW28 swing bucket rotor (Beckman Coulter, USA) spinning at 87300 × g for 22 h (Supplementary Figure S1). After the overnight centrifugation, the PM was extracted and underwent a fast centrifugation in an Avanti J-26S XP Centrifuge with JA25.50 rotor (Beckman Coulter, USA) at 27216 × g for 1 h to remove any residual sucrose. The PM pellet was then resuspended in water and gently sonicated using a Q700 sonicator (QSonica, USA). The concentration of PM was subsequently estimated with steady-state spectroscopy, using the absorption at 568 nm and the extinction coefficient of bR ε₅₆₈ = 62700 M⁻¹ cm^{−135 (link)}. The PM was then stored at 4 °C.

Translocation assays were performed as previously described (Baruch et al., 2011 (link)). Briefly, 3 × 10⁶ HeLa cells were infected for 3 h at a multiplicity of infection (MOI) of ~50 with EPEC strains that were grown statically overnight. The infected cells were then washed in cold PBS, collected, and lysed in designated lysis buffer [PBS with 0.5% Triton X-100 (v/v), protease inhibitor, and 1 mM DTT]. Thereafter, samples were centrifuged at 18,000 × g for 2 min to remove non-lysed cells, and supernatants were collected, boiled with SDS-PAGE sample buffer, and subjected to western blot with anti-Tir and anti-actin (loading control) antibodies. Uninfected samples and samples infected with the Δtir* mutant strain were used as negative controls.
To purify the cellular membranes of infected cells, washed cells were collected by scraping the plates. The cells were then resuspended in lysis buffer (3 mM imidazole [pH 7.4], 250 mM sucrose, 0.5 mM EDTA, and protease inhibitor) and mechanically fractionated by vigorous passage through a 25-G needle. The cellular lysates were centrifuged (3,000 × g for 15 min) to remove non-lysed cells and then further centrifuged (in a Beckman Optima XE-90 Ultracentrifuge with an SW60 Ti rotor at 100,000 × g for 40 min) to separate the cytoplasmic and membrane fractions.

For the production of AAV vectors, we used the triple transfection method. Fifty to seventy percent confluent AAVpro 293T cells grown in DMEM supplemented with 10% FBS were transfected with pHelper plasmid (20 mg/dish), pRC-DJ plasmid (11 mg/dish), and pAAV-IQSEC2 plasmid (10 mg/dish) using acidified PEI in 5 15-cm dishes. Two days after transfection, cells were collected and centrifuged at 1000 rpm for 5 min. The supernatant was removed, and the cells were resuspended in PBS and subjected to a freeze-thaw cycle 3 times. Following the addition of Benzonase (SIGMA-Aldrich), the cell lysate was incubated at 37 °C and centrifuged at 3600 rpm for 15 min. The supernatant was further purified using discontinuous iodixanol gradient ultracentrifugation using Optima XE-90 Ultracentrifuge (BECKMAN COULTER). AAV solutions were titrated by real-time quantitative PCR (Applied Biosystems) using primers KT18345/KT99100 for EGFP, KT19385/KT19363 for the hSyn promoter, or EFS1-F/EFS1-R for EFS promoter. Titers are expressed as viral genomes per mL (vg/mL). The titers for each AAV were 1.13 × 10¹¹ (vg/mL)(pAAV-EFS-Flag-IQSEC2), 1.40 × 10¹¹ (vg/mL)(pAAV- EFS), and 0.9 × 10¹⁰ (vg/mL)(pAAV-H1/U6-hSyn-EGFP).

For whole-genome sequencing, the clarified supernatants containing ASFV, for both the WSL-grown and the blood macrophage-grown virus stocks as described in the previous section, were further purified using a 36% sucrose solution. The supernatant was transferred into autoclaved, 250 mL, flat-bottom ultracentrifuge tubes, followed by centrifugation at 18,500× g for 2 h at 4 °C to pellet the virus particles in a Beckman Coulter Avanti Centrifuge J-301. The pellet was resuspended in 3 mL of 10 mM Tris (pH9), and the virus suspension was layered on 36% sucrose solution and subjected to ultracentrifugation at 30,000× g for 2 h at 4 °C, using the Beckman Coulter Optima XE-90 ultracentrifuge. The pellet containing the purified virus was resuspended in 10 mM Tris (pH9) and aliquots were stored at −80 °C.