Flow cytometric quantitation of EpCAM‐positive extracellular vesicles by immunomagnetic separation and phospholipid staining method

Extracellular vesicles (EV) have attracted attention as circulating biomarkers for many diseases, particularly cancer. Conventional immunofluorescence staining has been used for the detection of target antigens on EV by flow cytometry. However, the staining intensity depends on the amount of antigen expressed on the vesicles and is often only around the noise level. Instead of immunofluorescence, we combined immunomagnetic separation using nanosize MACS® MicroBeads with phospholipid staining of EV (IMS‐PS method). EpCAM‐positive EV were prepared from the culture supernatants of OVCAR3 (EpCAM‐high), A431 (EpCAM‐low) or Colon‐26 (non‐human control) cells as cancer models and were examined by the IMS‐PS method using EpCAM mAb‐coated MicroBeads. By employing Polaric‐500c6F as the dye for staining EV phospholipids and using appropriate flow cytometry settings, autofluorescence was excluded, whereas pretreatment of the MicroBeads with conventional blocking agents reduced nonspecific binding to non‐target vesicles. These modifications resulted in a linear relation between the number of EV detected and the sample volume, regardless of the level of EpCAM expression on the vesicles. A431 EV spiked into healthy volunteer plasma were enumerated with good accuracy. The IMS‐PS method may be useful for clinical evaluation of EV with low levels of antigen expression that are difficult to detect by conventional immunofluorescence. Extracellular vesicles (EV) in cultured cell supernatant are quantitated by immunomagnetic separation and phospholipid staining (IMS‐PS) method developed in the paper. Several improvements are made (A), appropriate amount of the bead is determined (B), and titrations of OVCAR3 EV and A431 EV show linear response between input EV sample volume and output counts (C). Colon‐26 EV are non‐human control.