A Systematic Approach to Improve Scatter Sensitivity of a Flow Cytometer for Detection of Extracellular Vesicles

Rond, Leonie de; Pol, Edwin van der; Bloemen, Paul R.; Broeck, Tina Van Den; Monheim, Ludo; Nieuwland, Rienk; Leeuwen, Ton G. van; Coumans, F.A.W.

doi:10.1002/cyto.a.23974

Cited by 20 publications

(35 citation statements)

References 31 publications

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“…First, we characterized EV preparations according to most recent guidelines. [ 30,31 ] SrtA‐3M‐TM+ EVs were produced at similar yields and had similar size as compared to control EVs (Table S2, Supporting Information, Figure 4 A). Both total protein and nucleic acid cargos of EVs did not significantly change when SrtA‐3M‐TM is present (Table S2, Supporting Information).…”

Section: Resultsmentioning

confidence: 89%

Cell Surface Labeling by Engineered Extracellular Vesicles

et al. 2020

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Deoxycytidine [3]). Extracellular vesicles (EVs) are increasingly recognized as a new class of multivalent messengers involved in cell-cell communication, both locally and at distance. [4-7] EVs are membranebound particles released by virtually every cell type, with stressed and transformed cells secreting the highest amounts. [8-11] Investigations of EVs' roles in different pathophysiological conditions are thwarted by biases introduced during isolation and reinfusion procedures. In particular, these biases manifest at different levels: (i) certain EV subsets may be excluded, [6] (ii) operator judgment is involved in deciding how many EVs to reinfuse, and (iii) an assumption that EV concentration is highest in blood, whereas several studies have shown that EV biodistribution occurs primarily via lymph fluid. [12-15] To circumvent these issues, the EV-releasing cells of interest can be genetically engineered to express membrane-bound reporters (or other functional biomolecules) in vitro or in vivo. [16] Although this approach is still in its infancy, it avoids the above limitations and to unbiasedly investigate untouched, endogenously released EVs. Although a detailed description of how EVs signal between cells is still missing, two nonexclusive mechanisms have been proposed: (i) membrane fusion between EVs and target cells, thereby releasing EV cargo (e.g., microRNAs and proteins, including oncogenes) directly in the cytoplasm of targeted cells; and (ii) surface binding with subsequent ligand-receptor type signaling. Membrane fusion (also known as horizontal transfer) has been described using several in vitro models (see Pucci and Pittet [17] and references therein), but proved more challenging to demonstrate in vivo. Recently, horizontal transfer of tumor-derived EVs was tested in mice using a sensitive and stringent approach based on the CRE-Lox reporter system. [15,18] Although CRE recombinase has an efficiency of 55%, [19] horizontal transfer was shown to happen mainly between tumor cells in vivo, but not between tumor cells and host cells. [15,18] These results have been further confirmed using other genetic strategies, [20,21] suggesting that surface binding may represent the main mechanism of EV-mediated tumor-host communications in vivo. A major limitation in the study of native EV signaling to host cells in vivo derives from their small size, which restricts the amount of reporter molecules carried by a single EV. As a consequence, only target cells binding the highest levels of EVs [12,14,15] will accumulate enough reporter molecules to be detected and investigated. Nonetheless, many host cell types Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically eng...

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Section: Resultsmentioning

confidence: 89%

Cell Surface Labeling by Engineered Extracellular Vesicles

et al. 2020

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“…Current EV quantification and characterization approaches are often limited by efficient detection of primarily larger vesicles. For example, NTA has an estimated limit for efficient detection of EVs of 60-70 nm 31 while dedicated highresolution flow cytometry and imaging flow cytometry are able to efficiently detect EVs as small as 60-100 nm 27,28,30,[32][33][34][35] . EVQuant size analysis is based on 3D intensity analysis of individual particles in the z-stack.…”

Section: Discussionmentioning

confidence: 99%

EVQuant; high-throughput quantification and characterization of extracellular vesicle (sub)populations

Hartjes

Slotman

Vredenbregt

et al. 2020

Preprint

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Extracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

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“…The side scatter (SSC) signal of the bead mixture was measured at illumination powers ranging from 20 mW to 200 mW for the 488 nm laser on a customized FACSCanto ( 10 ) (Becton Dickinson, Franklin Lakes, NJ). SSC collected on the customized FACSCanto is split by a dichroic mirror (NFD01‐488‐25x36; Semrock, Rochester, NY) and simultaneously detected by the standard SSC detection module and a high‐resolution SSC module (Supporting information Fig.…”

Section: Methodsmentioning

confidence: 99%