Shandon cytospin cytocentrifuge

Samples of 20 μL of exosomes were mixed with 1·5 mL of Diluent C and 4 μL of the red membrane dye PKH26 (Sigma-Aldrich, St Louis, MO, USA) and incubated for 10 min. The labelled exosomes were washed twice with PBS at 100 000 g; PBS from the top of the washed labelled exosome tube was used as the control. Thereafter, 5 × 10⁵ normal peripheral blood mononuclear cells (NPBMCs) or MyLa cells were incubated with labelled exosomes (15 μL) for 24 h. Target cells that internalized the exosomes were analysed by two methods. The first method was fluorescence microscopy. Cells were centrifuged with the Shandon Cytospin^® cytocentrifuge (Thermo Fisher Scientific Inc., Waltham, MA, USA), fixed with 4% paraformaldehyde, washed with PBS, and covered with a mounting medium containing 4′,6-diamidino-2-phenylindole (BioLegend, San Diego, CA, USA). Images were taken with an Axio Imager Z2 microscope (magnitude × 40; Zeiss, Jena, Germany). The second method was fluorescence-activated cell sorting (FACS). Cells were washed with PBS and analysed for PKH26-positive cells by FACS (Gallios™; Beckman Coulter, Brea, CA, USA).