Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?

Abstract

Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate; both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.

Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses

“…A large proportion of EVs cannot be included in the quantification due to their size and the limit of detection of the flow cytometer. Conventional flow cytometers are capable of detecting EVs of 100 nm in diameter or greater, thus excluding all smaller EVs [37]. In this study, EVs between 100 and 900 nm were analysed.…”

Microvesicles Transfer Mitochondria and Increase Mitochondrial Function in Brain Endothelial Cells

“…Fluorescent sub-micron size reference beads with mean diameters of 20-, 100-, 200-, and 500-nm were used to generate a size reference scale. Gating was applied to exclude the background laser noise in the scatter plots 599 [64]. The detection gate was set between 100 and 500 nm to allow the detection of the beads alone 600 (Fig.…”

Microglia and perivascular macrophages act as antigen presenting cells to promote CD8 T cell infiltration of the brain

“…This correlates with our DLS data wherein the mean effective particle size 608 diameters of D3-EXO and D3-MVs ranged from 160 -250 nm. Lucchetti et al also reported 100 609 nm-sized EXOs with a refractive index less than 1.4 based on the side scatter signal intensity 610 compared to the 100 nm polystyrene beads (refractive index of ~1.6) [64]. 611…”

“…Derivatives of calcein especially its acetomethoxy derivative (Calcein-AM) have been extensively studied for live cell tracking and detection. This has also been applied to distinguish functional and metabolically-active EVs from the disrupted EV or protein debris [64]. Fluorescent sub-micron size reference beads with mean diameters of 20-, 100-, 200-, and 500-nm were used to generate a size reference scale.…”

“…Fluorescent sub-micron size reference beads with mean diameters of 20-, 100-, 200-, and 500-nm were used to generate a size reference scale. Gating was applied to exclude the background laser noise in the scatter plots [64]. The detection gate was set between 100 and 500 nm to allow the detection of the beads alone 600 (Fig.…”