Jsm 7600f sem

The substrates with cells grown were washed with PBS and fixed in a mixture of 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, USA) and 2% glutaraldehyde (Fisher Scientific, Pittsburgh, PA, USA) in PBS at ambient for 4 h. The samples were then rinsed with deionized water and progressively dehydrated using a Tousimis Autosamdri-815 CO₂ Critical Point Dryer (Tousimis Co., Rockville, MD, USA).
The substrates with and without cells grown were sputter-coated with a gold layer of ~10 nm thick by using a Denton Vacuum Desk V sputter unit (Denton Vacuum, LLC, Moorestown, NJ, USA), and imaged in a JEOL JSM-7600F SEM.

Pellets containing extracellular vesicles isolated from healthy cells (i.e., exosomes) and from apoptotic and necrotic cells were vortexed and resuspended in 0.2-1 ml DPBS. Exosomes and nano-scaled apoptotic vesicles or necrotic bodies (microscaled parts of apoptotic vesicles or necrotic bodies were removed prior to ultracentrifugation) were fixed in a 2% EMS-quality paraformaldehyde aqueous solution. The samples were then diluted in distilled (dl) water in serial dilutions, added in 1-5 μl vesicle mixtures to cleaned silicon chips, which were sonicated in acetone, ethanol and distilled water for 5 min in each solvent, flushed by water and blown dry, and immobilized after drying vesicles under a ventilation hood. Samples on silicon chips were mounted on a SEM stage by carbon paste. To make surface conductive, a coating of 2-5 nm gold-palladium alloy was applied by sputtering (SPI-Module Sputtering, Argon as gas for plasma) before imaging by scanning electron microscopy Hitachi S-4700 or a JEOL JSM-7600F SEM. SEM was performed under low beam energies (5.0-10.0 kV). For best vesicle morphology under SEM, fresh isolated exosomes were fixed and immobilized on silicon right after isolation, and imaged within 7 days. Analysis of exosome sizes were done using the SEM images via ImageJ and the density distribution of exosome diameters were obtained using R/Bioconductor.