An Imaging Flow Cytometry-Based Methodology for the Analysis of Single Extracellular Vesicles 3 53 tetraspanin mixture similar to ours, has recently been published 33. In this study, membrane fluorescence was calibrated in terms of vesicle size (surface area) by using a synthetic vesicle size standard, as provided in the vFC EV Analysis kit from Cellarcus Biosciences. However, the staining with a lipid membrane dye should be consistent for applicability. Thus, either the amount of dye needs to be approximately matched to the number of EV, or an excess of dye should be used so that the membrane becomes saturated with dye 32. Additionally, the staining of lipoproteins is unavoidable when performing lipid staining strategies on PPP samples. It must be noted that the identification and quantification of single EV through the IFCM method presented here is also subject to limitations. First, a minimum of 3 pixels is required before an event is recorded by IFCM as an object; fluorescent events not passing this threshold may consequently be missed. Second, our gating strategy excludes multiplets from analysis, and only single-spot fluorescent events are quantified. This may yield underestimations of EV concentration in very concentrated samples (as the frequency of multiplets may be higher than that of singlets during the acquisition of such samples) 21. In such cases, serial dilution experiments may prove valuable to reduce multiplet detection and obtain a high frequency of single events. Alternatively, our gating strategy could be expended upon: rather than excluding events representing multiplets, the individual particles might be quantified and – following multiplication of the obtained concentrations with a factor representing their identified multiplet value – added to the total obtained concentrations of singlets. Combined, we propose five criteria for the successful analysis of single EVs in PPP through IFCM: 1) standardization of SSC signal intensities to allow estimation of particle sizes; 2) single-spot fluorescence to ensure single-particle analysis and no coincident events; 3) colocalization of a minimum of two fluorophores to assess the presence of two markers in the same particle or event; 4) disappearance after detergent treatment to confirm that the detected events represent structures composed of lipid membranes and hence are of biological origin; and 5) a linear correlation between concentration and dilution factor to further imply that single EVs are analyzed. These criteria are summarized in Table 1 for quick reference. In conclusion, we present an IFCM-based methodology and provide a framework that will allow researchers to directly study plasma-derived EVs, expanding on the usage of EV as non-invasive biomarkers in the clinic. We expect that this
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