Extracellular vesicles are a heterogeneous cell-derived particle population in a size range between 50nm to 1,000nm. There is a growing interest not only from academic research groups to determine EV in several fluids such as cell culture supernatant, in plasma samples or in whole blood but also in clinical research since it has been shown that the measurement of microparticles (MPs)1 might be of clinical relevance. The methods to identify EV are many and involve high speed centrifugation, Western blotting, proteomics, electron microscopy, imaging methods and flow cytometry. Methods for the detection of EV by flow cytometry have been developed in the last years and special attention has been paid to standardization protocols. Compared with other methods, flow cytometry has the big advantage that EV can be detected as rare events, in high numbers and by antigens on the surface, which characterize their cellular origin.
However, until now flow cytometry technology has had some unfortunate limitations. It was not possible to detect microparticles below 250-300nm in size in a meaningful manner. This size range does not appear to be very far from the smallest particles of 50nm in size, however we have to consider that MPs in a size greater than 300nm are only the “tip of the iceberg“ of visible particles and at least as many particles are smaller than 300nm in size. The importance in clinical research and the technical requirements to detect smaller microparticles was clearly demonstrated in 2013 by Sarlon-Bartoli et al2. Using a Beckman Coulter Gallios* flow cytometer they correlated an increase in plasma levels of leukocyte-derived MPs with unstable plaque in asymptomatic patients with high-grade carotid stenosis. These differences between sample groups were detectable using the Gallios flow cytometer which allowed for better discrimination between noise in an acceptable range and extracellular particles. The CytoFLEX is the first flow cytometer which can detect EVs in a meaningful way down to 150nm and therefore offers the possibility to detect particles below 300nm whichenhances information. The better resolution of the CytoFLEX can be reached by using the side scatter of the 405nm laser and by several technical as well as preanalytical preparations. Here we describe how to set-up and standardize the CytoFLEX for particle measurement and discuss some pitfalls which should be avoided to get the best information from EVs detection.