Deriving Extracellular Vesicle Size From Scatter Intensities Measured by Flow Cytometry

Abstract

Flow cytometry is commonly used to investigate the potential for extracellular vesicles (EVs) to be biomarkers of disease. A typical flow cytometer detects fluorescence and scatter intensities of single EVs in arbitrary units. These arbitrary units complicate data interpretation and data comparison between different flow cytometers. For example, comparison of detected EV concentrations requires knowledge of the detectable EV sizes. Using Mie theory and knowledge of the optical configuration of the flow cytometer, EV size can be derived from the scatter intensity for a given EV refractive index. Here, a protocol is described to derive the size of EVs and other nanoparticles from the scatter intensity. The resulting size distribution allows the comparison of data between flow cytometers, which is a prerequisite for clinical application of EVs as biomarkers and may advance other fields where sizing of nanoparticles is essential. © 2018 by John Wiley & Sons, Inc.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…FCM provides data in arbitrary units. To improve data interpretation and enable data comparison, fluorescence signals can be calibrated in units of molecules of equivalent soluble fluorophore (MESF) or equivalent reference fluorophore (ERF) and scatter signals can be calibrated in units of nm 2 . Do not use the scatter intensity of two sizes of polystyrene particles to set gates for two reasons.…”

Refractive index to evaluate staining specificity of extracellular vesicles by flow cytometry

“…All samples were measured for 2 min at a flow rate of 3.0 µL/min using SSC triggering (405-nm laser, 100 mW). The detection threshold was set at 14 a.u., which coincides with a scattering cross section of 5.3 nm 2 according to [21]. Isotype controls (Appendix, Figure A1) were used to set a fluorescent gate for CD61-PE, resulting in a gate with a lower boundary at 67 MESF of PE.…”

Extracellular Vesicles in Feto–Maternal Crosstalk and Pregnancy Disorders

“…It also has been purposed to stain EV samples with phalloidin, in order to exclude damaged EVs from the flow cytometry analysis, allowing a more accurate discrimination of intact vesicles [6,19,20,46]. Finally, the use of the Rosetta Calibration system, which permits the identification of the EV compartment on the basis of the particle size values, combined to a flow cytometry standardized procedure, significantly enhance flow cytometry sensitivity [47,48].…”