Quorum t150es sputter coater

The size of the CIMVs was determined by flow cytometry (BD FACSAria III, BD Biosciences, San Jose, CA, USA) with using a mixture of calibration particles (0.22–0.45–0.88–1.34 μm) (Spherotech, Lake Forest, IL, USA). In all the experiments with the CIMVs, a violet laser (Ex = 405, Em = 450) was used to detect particles from 200 nm in diameter. A minimum of 50,000 events were acquired for each sample. For sizing the CIMVs by scanning electron microscopy (SEM) analysis, CIMVs were isolated as previously described. Isolated CIMVs were resuspended in PBS and applied on glass slides by centrifugation at 3000 rpm for 30 min at room temperature. The CIMVs were fixed with 10% formalin for 15 min, dehydrated through an ethanol gradient from 30% to absolute and air-dried for 24 h. Prior to imaging, samples were coated with gold/palladium in a Quorum T150ES sputter coater (Quorum Technologies Ltd., Laughton, UK) and viewed for analysis by an SEM Merlin (Carl Zeiss, Jena, Germany). Two biological replicates were completed for the experiment.

The CIMVs were isolated from native MSCs, MSCs-BFP, and MSCs-TRAIL, as previously described, and analyzed using flow cytometer BD FACSAria III (BD Biosciences, San Jose, CA, USA). The size of the vesicles was determined comparatively to a mixture of calibration particles (0.22–0.45–0.88–1.34 μm) (Spherotech, Lake Forest, IL, USA). In all of the experiments with the CIMVs, a violet laser (Ex = 405, Em = 450) was used to detect particles from 200 nm in diameter. For sizing the CIMVs by scanning electron microscopy (SEM), the CIMVs were isolated as previously described, resuspended in PBS and applied on glass slides by centrifugation at 3000× rpm for 30 min at room temperature. The CIMVs were fixed with 10% formalin for 15 min, dehydrated through an ethanol gradient from 30% to absolute and air-dried for 24 h. Prior to imaging, the samples were coated with gold/palladium in a Quorum T150ES sputter coater (Quorum Technologies Ltd., Laughton, UK) and viewed for analysis by an SEM Merlin (Carl Zeiss, Jena, Germany). Two biological replicates were completed for the experiment.

CIMVs-MSCs and MSCs-derived EVs were fixed (10% formalin for 15 min), dehydrated using graded alcohol series and dried at 37 °C. Prior to imaging, samples were coated with gold/palladium in a Quorum T150ES sputter coater (Quorum Technologies Ltd, United Kingdom). Slides were analyzed using Merlin field emission scanning electron microscope (Carl Zeiss, Germany). For size analysis, three independent batches of CIMVs were produced and used to generate at least six electron microscope images for each batch. Data collected was used to determine the CIMVs size.