Glass bottom chamber slides

Fluorescence deconvolved images were taken using the DeltaVision Microscope system (GE Healthcare Life Sciences, Piscataway, NJ, USA). EVs were observed using glass bottom chamber slides (80807, Ibidi). Isolated EVs were incubated with 0.8 µg/mL APC-labeled annexin A5 (640919, BioLegend, San Diego, CA, USA) and 4.8 µg/mL FITC-GlaS for 30 min at room temperature and directly observed by fluorescence microscopy. The fluorescence filter set FITC/Cy5 and the 60x oil immersion objective, 1.42 NA, were used to acquire images and process z-stacks (Optical section space: 0.2 µm, Number of optical sections: 30) for deconvolution. Maximum intensity projection images of z-stack were created using ImageJ software (Version 1.53a, NIH).

Human Embryonic Kidney (HEK293-T) cell were purchased from ATCC® (USA), and cultured in phenol red free DMEM media (Gibco, UK) supplemented with 10% FBS, with non-essential amino acids, 1% penicillin–streptomycin and 2 mM Glutamine. For live-cell imaging, cells were seeded in 8 well glass bottom chamber slides (Ibidi) at concentrations of 2.5 × 10⁴ cells per well, 1 day before transfection. Cells were transfected with Fugene-HD transfection reagent (Promega, UK), as detailed by the manufacturer. 100–200 ng of DNA in 10 µl serum free, phenol red free DMEM media, mixed with 0.25 to 0.4 µl of transfection reagent and left in the tube with transfection complex for 10 min at room temperature. Then the transfection complex was added into wells containing cells, gently shaken and placed in a 37 °C incubator for at least 24 h.