Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis

Abstract

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.

A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders

“…Consequently, only a few membrane proteins will be enriched on the EVs while others, although present at the cellular scale, are not detectable on the EVs generated (Jimenez et al., 2019 ; Jang et al., 2019 ). Although NDEVs detection was established using one of the most fluo‐sensitive instruments available on the market (George et al., 2021 ), another explanation could be the expression of antigen with an insufficient density to be detectable by FCM. The limited detection of some antigens on NDEVs might also potentially be due to the epitopic specificity of the selected clone.…”

Medium Extracellular Vesicles—A Qualitative and Quantitative Biomarker of Prostate Cancer

“…In the case of extracellular vesicles, ultracentrifugation is the isolation method necessary for the precise selection of the material, which allows for particle fraction even below 30 nm. Unfortunately, the procedure itself is not precise enough, and the magnitude of the centrifugal force exposes the sample to contamination and decay of the tested particles, prompting researchers to constantly search for better alternative methods [ 33 , 34 , 35 ].…”

Heparin-Functionalized Adsorbents Eliminate Central Effectors of Immunothrombosis, including Platelet Factor 4, High-Mobility Group Box 1 Protein and Histones

“…Calibration of the flow cytometer was performed with fluorescent silica beads (1 µm, 0.5 µm, 0.3 µm, 0.1 µm; excitation/emission 485/510 nm; Kisker Biotech, Steinfurt, Germany). The triggering signal for EVs was set to the violet side scatter (405 nm), and the EV gate was set below the 1 µm bead cloud as previously described [14,69,70]. For platelet characterization, the triggering signal was set to the 488 nm side scatter (Supplementary Figure S2).…”