Fig. 2: Analysis and sorting of single cells based on EV secretion using nanovials and flow cytometry.

A Schematic of EV biogenesis and expression of tetraspanins (CD63, CD9, CD81) on the surface of EVs. B Tetraspanin abundance for EVs secreted by immortalized mesenchymal stem cells (iMSC) analyzed by ExoView. C Validation of sandwich immunoassay for detecting secreted EVs on nanovials. EVs were captured on anti-CD63 labeled nanovials and labeled with fluorescent anti-CD9 antibodies as shown in fluorescence and brightfield microscopic images. Scale bars represent 100 μm. D Analysis of EV secretion signal on nanovials loaded with cells using ImageStream imaging flow cytometry. A secretion signal mask was created by subtracting the calcein AM signal mask from the CD9 signal mask to exclude non-specific CD9 signals from cell surface staining. Scale bars represent 20 μm. E EV secretion+ population represents nanovials loaded with cells and containing high anti-CD9 signals spatially located across the cavity from captured EVs. F Differences in the ratio between the area of secretion signal and overall fluorescence intensity between high and low EV secretors result from spatially distributed secretion signals around the entire surface of the cavity. G Schematic of an experiment to evaluate crosstalk between cell-loaded and empty nanovials. Less than 1% of empty nanovials were observed to have an EV secretion signal when cell-loaded and empty nanovials were maintained in co-culture for 24 h. Scale bars represent 100 μm. H Single cells on nanovials were sorted based on staining for calcein AM and CD9+ EV secretion signal into three categories of high, medium, and low secretors. Fluorescence microscopy images showing sorted populations with corresponding secretion quantity gates. Scale bars represent 100 μm. A, D created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.