Exosome Surface Marker Profiling by Flow Cytometry

Exosomes were conjugated to aldehyde/sulfate latex beads with a diameter of 4 μm (catalog no. A37304, Invitrogen) in PBS overnight at 4 °C [34 (link)]. The mixture was then subjected to immunostaining and flow cytometric analysis [10 (link)]. The exosome-latex beads or cells were incubated with biotinylated protein L (Piscataway, NJ) to detect surface CAR expression. After incubation, staining was performed for 30 min using PE-conjugated streptavidin (Biolegend, San Diego, CA, USA) and the excess dyes were then washed. The kit (eBioscience) was used for fixation/permeabilization before intracellular staining for 45 min. The assay was carried out using an Attune NxT flow cytometer (Thermo Fisher Scientific) and data were analyzed using FlowJo software (version 10.8). The corresponding IgG antibody was used as an isotype control.