Effective Visualization and Easy Tracking of Extracellular Vesicles in Glioma Cells

Abstract

Extracellular vesicles (EVs) are nano-sized, membrane-bound structures secreted by cells and play critical roles in mediating intercellular signaling. EVs based on their size as well as mechanisms of biosynthesis are categorized as either microvesicles (200–1000 nm) or exosomes (30–200 nm). The EVs carry several biomolecules like proteins, DNAs, RNAs, and lipids into other cells and modulate several cellular functions. Being of very small sizes, it is very challenging to analyze them using conventional microscopes. Here, we report a new method developed by us for visualizing EVs using simple immune-fluorescence based technique, wherein the isolated EVs can be stained with fluorescently tagged antibodies to proteins present in EVs. The stained EVs can then be analyzed by using either confocal or super-resolution microscopes. Our method detailed here is equally effective in staining proteins that are present inside the EVs as well as those localized to the membranes of vesicles. By employing unique staining strategies, we have minimized the background noise and thereby improved the signal strength in confocal microscope. Using electron microscopy, we have ascertained that the structural integrity of the labeled EVs is intact. More importantly, the labeling of EVs does not affect their functionality and their localization can be tracked after its uptake by recipient cells without resorting to any conventional reporter-based strategies or lipophilic dyes. In conclusion, the method described here is a simple, sensitive and efficient immune-fluorescence based method for visualization of molecules within the EVs.

Cargo and cell‐specific differences in extracellular vesicle populations identified by multiplexed immunofluorescent analysis

“…An advantage of the EV-MAC approach described here is the relative ease by which EVs can be spinnoculated onto coverslips and subsequently analysed using conventional immunofluorescent imaging techniques [29,31]. Notably, other studies have previously demonstrated that immunofluorescent interrogation of EV populations is possible [35][36][37][38][39][40][41][42][43]. However, the workflow we describe allows the heterogeneity of EV populations to be rapidly assessed by investigators without super-resolution or singlemolecule approaches described in prior studies.…”

Extracellular Vesicles isolated from Mesenchymal Stromal Cells Modulate CD4+ T Lymphocytes Toward a Regulatory Profile

“…PKH dyes have been widely used for labeling extracellular vesicles [47,48], however, their use has been controversial. Recent studies have shown that nanoparticles of dye can also be internalized [49,50], but in a lower extension as EVs-labeled particles [49].…”

Potential roles of extracellular vesicles in the pathophysiology, diagnosis, and treatment of autoimmune diseases

“…For studying EVs size and morphology, reliable methods are electron microscopy (EM) techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), Cryo-electron microscopy (Cryo-EM), and atomic force microscopy (AFM). Confocal fluorescence microscopy (CFM) is another advantageous technique for studying the interaction between EVs and cells [41][42][43]. This technique has resulted very convenient to study the internalization of EVs by recipient cells [42][43][44][45][46].…”

“…Confocal fluorescence microscopy (CFM) is another advantageous technique for studying the interaction between EVs and cells [41][42][43]. This technique has resulted very convenient to study the internalization of EVs by recipient cells [42][43][44][45][46]. Mrinali et al developed an innovative method that combines formalin with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to improving EV imaging in situ.…”