Carbon coated copper grid

After charging the carbon-coated copper grids (Sigma-Aldrich) under ultraviolet light for 5 min, purified L1:P18I10 VLPs were absorbed on grids for 1 min and rinsed 3 times by miliQ water. The L1:P18I10 VLPs purified by ultracentrifugation in PBS (pH = 7.4, 137 mM NaCl) were negative-stained with 2% phosphotungstic acid (PTA) at pH 7.0 (Sigma-Aldrich) for 1 min. The L1:P18I10 VLPs purified from chromatography in Tris-HCl (pH = 7.4, 137 mM NaCl) were negative-stained with 2% uranyl acetate at pH 4.5 (Sigma-Aldrich) for 1 min. Excess staining agents were removed by Whatman qualitative filter paper (Sigma-Aldrich). Grids were placed in a dehumidifier chamber at least 2 h before observation. Images were acquired using a transmission electron microscopy (Tecnai Spirit 120 kV, FEI Company, Hillsboro, OR, USA) at magnification SA270K (50 nm), SA59000 (200 nm) and SA529500 (400 nm), respectively.

After charging the carbon-coated copper grids (Sigma-Aldrich) under ultraviolet light for 5 min, commercial HPV16 L1 (Abcam), purified L1:P18I10 and L1:T20 VLPs equilibrated with 20 mM Tris-HCl (pH 7.4, 137 mM NaCl) were absorbed on grids for 1 min and rinsed three times by miliQ water. The HPV:HIV VLPs were negative-stained with 2% uranyl acetate at pH 4.5 (Sigma-Aldrich) for 1 min. Excess staining agents were removed by Whatman qualitative filter paper (Sigma-Aldrich). Grids were placed in a dehumidifier chamber at least 2 h before observation. Images were acquired using a transmission electron microscope (Tecnai Spirit 120 kV) at magnification SA135K (100 nm) and SA59000 (200 nm), respectively.

Bacteriophages were concentrated by centrifugation at 25,000 ×g for 60 minutes using a high-speed centrifuge. The purified phages were deposited on carbon-coated copper grids (Sigma-Aldrich, Germany) and stained with 2% uranyl acetate (pH=4-4.5). After staining, phages were observed using a Philips CM 300 electron microscope (Philips, USA) at 150 kV. ^{23 (link)
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To obtain M0-Exo or M1-Exo, the culture media of M0 macrophages or activated M1 macrophages were collected and centrifugated at 800×g, 3000×g, and 10,000×g for 10 min, 10 min, and 30 min, respectively, at 4 °C, to remove cell debris and large extracellular vesicles. Then the culture media were ultra-centrifugated at 100,000g with a type Ti41 rotor (Optima XPN-100, Beckman Coulter) for 70 min at 4 °C to obtain the M0-Exo or M1-Exo. The weight of exosomes was determined by a well-established quantification method of total protein using a Micro BCA Protein Assay kit. After constructing a protein standard curve (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 μg/μL) by measuring absorbance at 562 nm, the corresponding protein concentrations of different experimental groups were calculated.
The morphology of both M0-Exo and M1-Exo were characterized using a transmission electron microscope (TEM, Talos F200X). The isolated exosomes were diluted in PBS and 10 μL of M0-Exo or M1-Exo solution were dropped onto carbon-coated copper grids (Sigma-Aldrich). After drying for 5 min, 1% uranium acetate (Sigma-Aldrich) was added to stain the samples for 1 min. Samples were dried for another 20 min and examined by TEM. Hydrodynamic particle size was measured by dynamic light scattering (DLS, Nano-zs30, Malvern).