Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry

Abstract

The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform their use as biomarkers for disease.

Exercise-induced circulating extracellular vesicles protect against cardiac ischemia–reperfusion injury

“…Analysis of the nano-FCM results demonstrated the presence of a large population of EVs (Fig. 1a) with some variance of sizes at baseline as has been previously reported [14]. We observed a significant increase in the population of EVs in the gate based on forward and side scatter characteristics (normalized to standardized spike-in beads) and previously shown to correspond to EVs during peak exercise in patients compared to their resting state (43.11%±24.61% vs 54.65%±25.68%, P <0.05, paired t-test, 2 tailed) (Fig.…”

“…Plasma was processed and analyzed using nano-flow cytometry (nano-FCM) as previously described [14]. The mean age of the patients was 54±11 years and the mean Body Mass Index (BMI) was 27±4 kg/m 2 .…”

“…Photon Correlation Spectroscopy (PCS) latex beads (100 nm, 200 nm, 300 nm, and 500 nm; PN 6602336, Beckman Coulter, Fullerton, CA), and cell-free plasma were analyzed on the primarily AstriosEQ and also NanoView-equipped MoFlo XDP cell sorter as previously described using a 70 μm nozzle tip with sheath pressure set to 60 PSI [14]. AstriosEQ is equipped with two FSC PMT pathways separated by a beam splitter where FSC1 is a direct laser beam pathway and FSC2 is directed at an angle from the beam splitter.…”

Extracellular Microvesicle Production by Human Eosinophils Activated by “Inflammatory” Stimuli

“…EVs secreted by cells can be detected by nanoscale flow cytometric methods, which identify and sort submicron particles (Danielson et al, 2016) and transmission electron microscopy (TEM), which enables unambiguous visualization of EVs (reviewed in Lawson et al, 2016). EM is thus considered an essential technique to characterize EVs and to distinguish them from non-membranous particles of similar size, as endorsed by the International Society for EVs in an effort to provide minimal requirements for EV definition (Lötvall et al, 2014).…”

The Role of HTLV-1 in Alteration of Immune Regulation Through Regulatory T Cell Dysfunction and Exosomal Manipulation

“…4C, red bars). Samples were also analyzed by nanoFACS, a technique which quantifies vesicles in media by forward and side scatter (FSC, SSC) (Danielson et al, 2016). As seen in Fig.…”