Exo glow green labeling kit

EVs were isolated and stained using the Exo-Glow-Green labeling kit (System Biosciences) as per the manufacturer’s protocol. mMSC cells were plated onto 96 well cell culture plates at a concentration of 10,000 cells per well. The cells were then incubated with increasing amounts of fluorescently labeled EVs for 2 hours at 37°C, washed with PBS and fixed in 4% PFA. The fluorescence from the endocytosed EVs were observed and quantified by using a BioTek Synergy 2 96 well plate reader equipped with the appropriate filter sets to measure green fluorescence. The fluorescence intensities were normalized to background and no EV fluorescence as a function of dosage (n=6 per group). For qualitative endocytosis experiments, 50,000 mMSCs were plated on cover glasses placed in 12 well cell culture plates. Fluorescently labeled EVs (1.5×10⁹ particels) were added and incubated for 2 hours, washed, fixed, permeabilized and counter stained using Alexa Fluor® 568 Phalloidin (1/2000, A12390, Invitrogen) antibody. The cover glasses were then mounted using mounting medium with DAPI (Vector Laboratories) and imaged using a Zeiss LSM 710 Meta confocal microscope.

The exosomes were labeled using the Exo-Glow-Green labeling kit (System Biosciences) as per the manufacturer’s recommended protocol. During each labeling reaction, a control reaction was performed using PBS not containing exosomes. This mixture was used in subsequent experiments as control to ensure against non-specific staining from the labeling procedure.