Ti2 microscope

Widefield images were acquired using a Nikon Ti-2 microscope equipped with a 60 × 1.49NA objective and an ORCA-Flash 4.0 v3 sCMOS camera (Hamamatsu, Hamamatsu City, Japan). Images were deconvolved with Nikon Elements by the Richard-Lucy method for 20 iterations. dSTORM experiments were conducted on a Nikon Ti-2 N-STORM microscope equipped with a 100 × 1.49NA oil immersion objective, 488 and 647 nm lasers, and an iXon ultra EMCCD camera (Andor, Oxford Instruments). 60,000–80,000 frames were collected with subcritical inclined excitation and reconstructed in Nikon Elements. dSTORM imaging buffer included glucose oxidase (Sigma, St Louis, Missouri), glucose (Sigma), catalase (Roche, Penzberg, Germany), and β-mercaptoethanol (Sigma)^{52 , 53 (link)}. A detailed dSTORM protocol has been described previously.^{15 (link)}

Pluripotency of iPSCs was confirmed over multiple passages and during EV production via immunofluorescence imaging. Cells were fixed using a 4% paraformaldehyde and 1% sucrose solution for 15 minutes before washing three times with 1x PBS. The cells were then permeabilized using a 6 μM magnesium chloride, 20 μM HEPES, 100 μM sodium chloride, 300 μM sucrose and 0.5% Triton-X-100 solution for 5 minutes. After additional washing with 1x PBS, cells were stained with either Oct-4 (2890S, C52G3; Cell Signaling Technology Incorporated, Danvers, MA, USA) at a 1:500, or SSEA-4 (4755S, MC813; Cell Signaling Technology Incorporated, Danvers, MA, USA) at a 1:200 dilution and incubation overnight at 4°C. The following day, either a goat anti-rabbit (A32731; Thermo Scientific, Waltham, MA, USA) or goat anti-mouse (A32728; ThermoFisher Scientific, Waltham, MA, USA) secondary antibody at a concentration of 10 μg/mL was incubated on the cells for 1 hour in the dark. The cells were then stained with Hoechst 33342 (62248; ThermoFisher Scientific, Waltham, MA, USA) before imaging with a Nikon Ti2 microscope (Nikon; Minato City, Tokyo, Japan).