Formvar carbon coated 300 mesh copper electron microscopy grids

Exosomes were extracted from macrophages, as reported previously [22 (link)]. Briefly, macrophages were cultured in the exosome-depleted medium for 72 h. Then, the cell medium was harvested and centrifuged at 300 g for 10 min at 4°C and the media were filtered using a 0.22 μm filter. Finally, the media was ultracentrifuged at 120000 g for 70 min at 4°C and exosomes were collected. The exosomes were resuspended in 50 μl of PBS for the following experiments. Subsequently, the isolated exosomes were determined by transmission electron microscopy (TEM), particle analyzer, and specific protein markers. Exosomes were fixed with 1% buffered glutaraldehyde for 10 min. The 20 μL of exosomes were added to formvar carbon-coated 300-mesh copper electron microscopy grids (Agar Scientific Ltd., Stansted, UK) and allowed to stand for 5 min. Then 2% uranyl oxalate was added to counterstain exosomes at room temperature. After the grids were washed three times with PBS, the exosomes were photographed using TEM (Hitachi H7500 TEM, Tokyo, Japan). The exosome particle size analysis was conducted by nanoparticle tracking analysis (NTA) with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). The CD63 was applied to identify exosomes via western blot analysis.

Exosomes were extracted from BMSCs and purified by Total Exosome Isolation Reagent (4,478,359, Invitrogen, USA). Morphology of exosomes was identified by transmission electron microscopy (TEM). Briefly, exosomes were diluted to 500 μg/ml and fixed with 2.5% glutaraldehyde for 2 h. Approximate 10 μL of exosome suspension was placed on formvar carbon-coated 300-mesh copper electron microscopy grids (Agar Scientific Ltd., Stansted, UK), and incubated for 5 min at room temperature. The grids were washed three times with PBS, air dried for 5 min, and imaged under TEM (HT7700; HITACHI, Tokyo, Japan) at 80 kV. Nanoparticle tracking analysis (NTA) was detected by ZetaView PMX 110 (Particle Metrix, Germany). The diameter and concentration were calculated by Stocker-Einstein equation and the data was analyzed by Zetasizer software (Malvern Instruments). FLSs were cultured to 70% confluence on a sterile 6-well culture plate at 5 × 10⁵–1 × 10⁶ cells/well. The BMSCs exosomes were co-incubated with FLSs for 24 h.

Extracted exosomes were fixed in 1% dialdehyde for 10 min and washed with deionized water. In this study, 10 µl of exosome suspension was placed on formvar carbon-coated 300-mesh copper electron microscopy grids (Agar Scientific Ltd., Stansted, UK) and incubated at room temperature for 5 min. Then, the exosomes were negatively stained with 2% uranyl oxalate for 1 min at room temperature and followed by washing three times with PBS and drying at room temperature. Images were obtained by microscopy [transmission electron microscopy (TEM)] (JEM-2100, Quan Luo, Japan).