Ultracut uct ultramicrotome

Samples were prepared as previously described^{74 (link)}. In brief, PAO1 was incubated in CA-MHB overnight at 37 °C. After centrifugation, bacteria were resuspended in fresh CA-MHB containing NV716 at different concentrations during 1 h. Bacteria were fixed in 1% glutaraldehyde in 0.1 M phosphate buffer for 4 h. After post-fixation in 1% osmium tetroxide for 1 h and dehydration in ethanol, samples were embedded in resin. 70 nm sections were cut with a Leica ultracut UCT ultramicrotome and examined (unstained) in a JEOL 1400 transmission electron microscope equipped with an 11 Mpxl EMSIS Quemesa camera.

MSCs were cultured in Roswell Park Memorial Institute medium containing 5% EVs-depleted FBS (Systems BioSciences, Palo Alto, CA, USA) and 1% penicillin/streptomycin (Invitrogen). MSCs were seeded in a cell culture dish (100 mm) at a density of 2 × 10⁵ cells/dish and allowed to attach overnight. The medium was renewed for another 3 days of culture. When cell confluence reached approximately 80%, EVs were then detached from the conditioned medium using ExoQuick-TC EV purify reagent (Systems Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions. The obtained EVs were resuspended in phosphate buffer saline (PBS) (approximately 200 μL) and preserved at -80°C for subsequent quantification by Protein Assay bicinchoninic acid (BCA) Kit and for molecular analysis.
Transmission electron microscopy (TEM) was used to analyze EVs structure. EVs were fixed in 2% glutaraldehyde and 2% paraformaldehyde (PFA) in 0.1 M Sorensen’s phosphate buffer for 3 h, in 1% OsO₄ for 30 min, washed, dehydrated in graded series of 50%, 70%, 80%, 90%, and 100% ethanol and embedded in Epon 812. Ultrathin sections (60 nm) were cut with a Leica Ultracut UCT ultramicrotome, stained with uranyl acetate and Reynolds lead citrate, and observed under a TEM (JEOL JEM-1230). The size of EVs was measured using nanoparticle tracking analysis by means of NanoSight NS300 (Malvern, Amesbury, UK)