Slgpr33rb

In total, 30 rats were used to isolate serum exosomes for the whole study. About 7ml whole blood could be collected from one sham rat via the abdominal aorta into a vacutainer. The whole blood was allowed to clot at room temperature for 30 min. The serum (1–2 ml) was obtained by centrifugation at 2,500 g for 10 min. The serum was ultracentrifuged at 100,000 g for 60 min (Beckman Optima L-100XP, Beckman, United States) and the pellets were resuspended in phosphate-buffered saline (PBS) and then ultracentrifuged at 150,000 g for another 90 min. The pellets were resuspended in 1 ml PBS and passed through the 0.22 μm filter (Millipore, SLGPR33RB) to obtain sterile exosomes. The protein concentration of exosomes was determined with bicinchoninic acid (BCA) commercial kit (Vazyme, China) as manufacturer’s instruction. The protein concentration of 1 ml exosomes is 0.1–0.2 mg/μl. The exosomes were aliquoted and stored at –80°C until use.
The serum exosomes (labeled as Con-exo) were observed using transmission electron microscopy (TEM) to identify the morphology. The size and concentration distribution profile of serum exosomes were analyzed using a Multiple-Laser ZetaView^® f-NTA Nanoparticle Tracking Analyzers (Particle Metrix, Germany). The exosome surface markers were confirmed by Western blotting using antibodies against CD9 and TSG101.

The 1 mL plasma samples were filtered via a 0.22 μM filter (SLGPR33RB; Millipore). The supernatant was loaded into a Sepharose‐based CL‐2B column (Echo9101A‐5 mL; Echobiotech). The EVs particles were separated by PBS (P1020; Solarbio). Five hundred microliters of outflow was determined as a fraction. The 2−4 fractions were collected. Next, the effluents were ultracentrifuged at 150,000×g for 4 h. The pellet was resuspended by PBS and ultracentrifuged again at 150,000×g for 2 h. In the end, the EVs was resuspended with PBS.

PLA₂ and SVMP were purified from the crude venom of T. stejnegeri and P. mucrosquamatus, respectively, by fast protein liquid chromatography (FPLC) (AKTA pure, GE Healthcare, Boston, MA, USA) with a cation exchange column (Resource™ S, 6 ml, GE Healthcare, Boston, MA, USA), with elution flow rates of 2 and 3 ml/min, respectively. In brief, 50 mg of crude T. stejnegeri or P. mucrosquamatus venom was dissolved in 1 ml of buffer A (8 mM NaHPO₄·12H₂O, 1.5 mM KH₂PO₄, pH 7.4), then filtered through 0.22-μm sterile filters (SLGPR33RB, Millipore, Burlington, MA, USA), and loaded into the cation exchange column pre-equilibrated in buffer A. Subsequently, the column was washed with buffer A to elute non-binding proteins, and binding proteins were eluted by applying a salt gradient from 0 to 1 M NaCl in buffer A. Finally, the column was equilibrated with buffer A to prepare for the next sample load. During the elution process, each fraction was collected according to the absorbance peaks at 215 nm and lyophilized for further analysis.

HBV production and infection process were performed as described previously (51 (link)). HBV stable replication cell line HepAD38 was used to produce HBV virions. The medium containing HBV was first filtered through a 0.22 μm filter (Millipore, SLGPR33RB) and then concentrated with 100 kDa Ultra Centrifugal Filters (Millipore, UFC710008). For HBV infection, HepG2-hNTCP-K7 cells were seeded, pre-treated with culture medium containing 2.5% DMSO (Sigma-Aldrich, D2650) for 2 days prior to inoculation with about 200 HBV genome equivalents per cell (MOI 200) in the presence of 4% PEG8000 (Sigma-Aldrich, 89510). After 24 h, the infection inoculum was removed, and cells were washed three times with PBS (Gibco, C10010500BT), followed by routine maintenance.

HEK-293T cells were cultured in DMEM supplemented with 10% FBS, then transfected with HIV Gag/Pol, HIV rev, plenti-EGFP and the WT or mutated SARS-CoV-2 Spike expression plasmids using Lipofectamine 2000 reagent (Invitrogen, 11668019). The Spike-pseudotyped virus in supernatant was harvested at 48 h post transfection, carefully clarified by centrifugation at 1200 rpm for 10 min at 4 °C and then filtered through a 0.22 μm membrane (Millipore, SLGPR33RB). The virus was frozen at −80 °C.
ACE2-293T or Vero-E6 cells were seeded in 96-well plates and incubated with WT/mutated Spike pseudovirus for 48 h. The infection efficiency was recorded by fluorescence microscopy and positive clones were automatically quantified by Cytation 5.

Exosomes were isolated from plasma and supernatant as previously reported [19 (link)]. Briefly, samples were first filtered through a 0.22 μM filter (SLGPR33RB, Millipore, USA). Next, the filtrate was collected and added to an SEC column (Echo9101A-5 mL, Echobiotech, China) loaded with cross-linked agarose beads (commercially available as Sepharose® (CL2B300, Sigma Aldrich, USA) for splitting. Subsequently, 2–4 fractions were collected and centrifuged in 100 KD Amicon Ultra-15 ultrafiltration tubes (UFC810024, Millipore, USA). Finally, the obtained exosomes were resuspended in 800 μL Trizol reagent (15,596,018, Invitrogen, USA) to conduct the RNA extraction operation.