Abstract
Extracellular vesicles (EVs) are heterogeneous in size (30 nm–10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring.
Highlights
The term extracellular vesicle (EV) refers to particles released from cells which are delimited by a lipid bilayer, but do not contain a nucleus [1]
Large EVs from the medulloblastoma cell line UW228-2 are visible with light microscopy, can be isolated from viable, serum-free cell culture supernatant with intact membranes, and contain a polymerised actin cytoskeleton
Isolated EVs were spiked with medulloblastoma parent cells for comparison and adhered to ACLAR film coated with CellTak prior to transmission electron microscopy (TEM)
Summary
The term extracellular vesicle (EV) refers to particles released from cells which are delimited by a lipid bilayer, but do not contain a nucleus [1]. EVs are heterogeneous in biogenesis [2], size, and content They range in size from 30 nm exosomes [3] to oncosomes up to 10 μM [4], and contain cargo of all biomolecule categories [5]. Multiple commercial solutions exist for the isolation of exosomes from a variety of biological fluids including tissue culture supernatant, plasma, and urine. Large EVs, defined as >200 nm by recent guidelines set out by the International Society of Extracellular Vesicles [1], include cancer cell-derived oncosomes, dead cell-derived apoptotic bodies and platelets, and are visible by light microscopy [9]. We and others [4] have demonstrated that viable cell cultures produce large EVs which do not have the ultrastructural features reminiscent of fragments of apoptotic cells
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