Multiplexed profiling of SCC25 single-cell EV secretion revealed previously unobserved secretion heterogeneity. (A) Representative images showing the raw data of multiplexed single-cell EV profiling, including fluorescence detection images (partial and enlarged), and corresponding cells in microchambers (red circled are single cells). (B) Scatter plots showing five-plexed EV secretion profiling from SCC25 single cells (n = 1,264 and 1,417 for zero cells and single cells, respectively). The fluorescence intensity and secretion frequency were normalized with the average plus 2 SD of the zero-cell data. **P < 0.005; ***P < 0.0005; ****P < 0.00005; ns, not significant (P > 0.05). (C) Histograms showing secretion intensity distribution of different EVs (CD63+EV, CD81+CD63+EV, and CD9+CD63+EV). (D) Polyfunctionality analysis of SCC25 single cells (fraction of cells to secrete multiple EVs simultaneously). (E) Scatter plots showing the change of CD9+CD63+EV secretion frequency with the increased number of cells per microchamber. (F) Heatmap showing EV−EV correlation in single cells.