Enhanced flow cytometry-based EV profiling using lipophilic dyes and immunocapture techniques

Flow cytometry analysis of extracellular vesicles (EVs), including exosomes, remains challenging due to their small size and heterogeneity. This application note presents an optimised workflow combining fluorescent lipid membrane labelling with immunocapture using anti‑tetraspanin magnetic beads to improve EV detection and phenotypic characterisation.

Developed in collaboration with Acoerela and Miltenyi Biotec, this approach enables sensitive and specific EV analysis using standard flow cytometry platforms.

Method highlights

• Combined use of fluorogenic lipid membrane dyes (Aco‑Dyes™) and magnetic bead‑based immunocapture for EV detection by flow cytometry.
• Selection of low‑autofluorescence magnetic beads compatible with multicolour analysis.
• Optimised EV labelling using the Aco‑430 dye, providing strong fluorescence intensity and specificity.
• Capture of heterogeneous EV populations using mixed anti‑CD9, CD63 and CD81 antibody‑coated beads.

How this supports your extracellular vesicles characterisation programme?

This workflow supports robust and reproducible EV analysis by addressing key limitations of small particle detection.

• Improves sensitivity and specificity of EV detection in complex samples.
• Enables phenotypic characterisation of EV surface markers by flow cytometry.
• Reduces background signal through optimised washing strategies without mandatory ultracentrifugation.
• Facilitates analysis of EV populations with variable tetraspanin expression.

Funding and acknowledgements

This research is funded by the Walloon Region as part of the PIT ATMP Project – Project 42, Walloon Recovery Plan (PIT ATMP – Convention 8880 – Gene Therapy).

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