0.45 μm filter

Vesicular stomatitis virus (VSV)-G protein pseudotyped lentiviruses and retroviruses were prepared by using polyethylenimine (PEI; Sigma) to co-transfect 293T cells with a lentiviral or retroviral plasmid and pantropic VSVg (Addgene) and either psPAX2 (Addgene) (lentivirus) or pCL-Eco (retrovirus) packaging plasmids. After culture in HMEC-1 medium for 48 hrs at 37°C, the viral supernatant was harvested, filtered through a 0.45 μm filter (Thermo Fisher Scientific), and used immediately. Cells stably expressing pHAGE-xCT-HA-FLAG were selected in medium containing 1 μg/mL puromycin dihydrochloride (Corning), and cells expressing pMXS-SLC1A3 were selected in 5 μg/mL blasticidin (Invivogen).

Proteins were expressed using the Expi293™ expression system (Thermo Fisher Scientific, Waltham, MA, USA), which is a high-yield transient expression system based on suspension-adapted human embryonic kidney (HEK) cells. In brief, for the scalable transfection of the Expi293F™ cell lines in the Expi293™ expression medium (Gibco, Grand Island, NY, USA), 30 μg of plasmid was transfected into 30mL Expi293F cells at a cell density of approximately 4.5–5.5 × 10⁶ viable cells/mL. The cells were incubated at 37 °C with a humidified atmosphere of 8% CO₂ on an orbital shaker. After 3 days, the cell supernatant was collected. The supernatant was centrifuged at 3000g for 10 min and filtered through a 0.45 μm filter (Thermo Fisher Scientific). All purified proteins were concentrated using a 30kDa ultrafiltration tube (Millipore, Bedford, MA, USA), and the buffer was replaced with PBS. The concentration of the protein was measured using a BCA kit (Pierce™ BCA Protein Assay kit, Thermo Fisher Scientific). Proteins were characterized using reduced and non-reduced SDS-PAGE [43 (link)].

EV isolations from the brains were carried out as described previously with modifications according to the protocol [22 (link)]. The fresh and previously frozen mice hemibrains were harvested and dissected finely. The brain samples were then treated with 20 units/ml papain (Worthington) in Hibernate E solution (BrainBits, Springfield, IL) for 15 min at 37 °C. The same volume of cold Hibernate E solution was added to the brain samples to stop the reaction of papain. The brain tissue was then gently homogenized and filtered through a 40-μm mesh filter (BD Biosciences), followed by a centrifugation at 300×g for 10 min and 3000×g for 20 min at 4 °C to get rid of cells, membranes, and debris. After the supernatants were filtered through 0.45-μm filter (Thermo Scientific), they were subjected to 10, 000×g for 30 min at 4 °C to eliminate organelle contaminations. The supernatants were further centrifuged at 100,000×g for 70 min at 4 °C to pellet EVs. The pellets were then resuspended in filtered PBS, or MPER lysate solution for NanoSight or Western blot. All the samples were ultracentrifuged in ultraclear polycarbonate tubes (Beckman Coulter) that have a volume of 13.2 ml. A Beckman Coulter ultracentrifuge (Beckman Coulter OptimaL-90K ultracentrifuge; Beckman Coulter, Fullerton, CA, USA) was used with a rotor type SW 41 Ti.

Porcine skin is a commonly employed alternative to human tissue in in vitro studies [3 ]. Full-thickness skin samples were mounted on glass Franz-type diffusion cells (n = 6 for each test), with diffusion areas of 3.14 cm² and a receptor volume of 15 mL. Solutions (3 mg/mL) of PMD and the copolymer were prepared in water/ethanol (8:2 v/v) and 1 mL (3 mg) was dosed into the donor chamber. The Franz cells were placed in an incubator at 32 ± 1 °C; samples (1 mL) were taken periodically from the receptor compartment between 0.25 and 72.00 h, and were replaced with equivolume of the water/ethanol (8:2 v/v) receptor medium.
Following the 72 hrs permeation study, PMD and copolymer remaining in the tissue was assayed by tape stripping. The first two tape strips were assumed to account for analyte remaining on the skin surface, tape strips 3–10 accounted for deposition in the upper stratum corneum, and strips 11–20 showed analyte remaining in the lower stratum corneum. To extract the PMD or copolymer, tape strips were immersed in ethanol and sonicated for 15 min at room temperature. Any residual tissue was removed by filtration (0.45 μm filter, Thermo Fisher, United States), and the extraction liquid analysed by gel permeation chromatography (GPC) for the copolymer and LC-MS for PMD.

The sequence of the extracellular region of G proteins from HeV and NiV (HNV-G) were codon-optimized, the tPA signal peptide and Strep-II tag were added at the N-terminus. Gene was synthesized (General Biosystems Co., Ltd.) and inserted into pcDNA3.1 (+).
The plasmid was transfected into Expi-293F cells for expression. After transfection, cells were placed on a shaker at 120 rpm and 37°C. After 72 hours of cell culture, the supernatant was centrifuged at 3,000 g for 15 min and filtered through a 0.45 μm filter (Thermo Fisher Scientific, USA). The protein was purified using the StrepTrap HP affinity column (GE Health Care, USA). Purified proteins were concentrated using 30 K ultrafiltration tubes (Merck Millipore, Germany) and permuted using PBS buffer at pH 7.4. Finally, the obtained proteins were characterized using SDS-PAGE and quantified with a BCA Protein Assay Kit (Thermo Fisher Scientific, USA).

The thawed dinoflagellates were centrifuged (300× g, 5 min, 25 °C) for removing the CPA. Fresh FSW was added for resuspending the samples. The samples were then transferred into a 24-well microplate (Cosmo Biosciences Inc., CA, U.S.A.) which was sealed and stored in a culture incubator set to a 12 h light:12 h dark cycle (Kansin Instruments CO., LTD.). The culture medium was prepared by mixing 32 g of Coralife Salt Water Mix (WI, U.S.A) with 1 L of distilled water to create 32 ppt artificial seawater. After the Salt Water Mix was fully dissolved, 30 mL of Guillard’s f/2 medium and 1 mL of an antibiotic solution were added to 970 mL of the artificial seawater. Both the antibiotics (penicillin: 100 units/ml; streptomycin: 10 mg/ml) and Guillard’s f/2 medium were purchased from Sigma-Aldrich (St. Louis, MO., U.S.A.). The solution was filtered using 0.45 μm filter (Thermo Scientific Inc., Massa., U.S.A.) after it had evenly dissolved. The culture was replenished with fresh medium every two days. During the medium exchange, 10 μL aliquot of the culture was retrieved for cell counts.