eoe sub articles

EoE Sub Articles

1. EV isolation and on-chip immuno-fluorescent EV labeling
<ol>
	<li>Collection of cell culture media (CCM) and pre-processing of CCM for EV isolation
	<ol>
		<li>Seed PC3 cells at 30% confluency in a 75 cm2 cell culture flask. Allow control cells to grow to 90% confluency (~48 h) in standard media and cell-line-specific supplements. 
		NOTE: To prevent EV-containing components from affecting cellular uptake (i.e., fetal bovine serum), use exosome-depleted media and supplements.</li>
		...

extracellular vesicle uptake assay: a method to visualize and quantify cellular uptake of extracellular vesicles using 3d fluorescence imaging technique

Source: Kim, C. J., et al., <a href="https://www.jove.com/v/62836">Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis.</a> J. Vis. Exp. (2022).
In this video, we demonstrate an assay to visualize and quantify the cellular uptake of extracellular vesicles or EVs. This assay utilizes 3D fluorescent imaging using a confocal microscope and allows for distinguishing internalized EVs from ones adhered to the cell surface.

<img alt="Figure 1" src="/files/ftp_upload/21154/21154_Figure1.jpg" /> 
Figure 1: Schematic illustration of the EV isolation and on-chip labeling using a nano-filtration-based microfluidic device.&#160;(A) EVs isolation from CCM. (B) On-chip Immunofluorescent labeling of EVs. (C) Removal of unbound antibodies.
<img alt="Figure 2" src="/file...

Extracellular Vesicle Uptake Assay: A Method to Visualize and Quantify Cellular Uptake of Extracellular Vesicles Using 3D Fluorescence Imaging Technique

Source: Kim, C. J., et al., Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis. J. Vis. Exp. (2022).
In this video, we ...

To facilitate cellular communication, cells secrete extracellular vesicles, EVs - nano-sized structures enclosed by a lipid membrane. EVs carry various biological cargoes, including macromolecules and metabolites, for delivering them to the recipient cells.
To visualize EV uptake by cells using confocal microscopy, first, take freshly isolated EVs. These EVs are pre-labeled with antibodies bound to a fluorescent probe for subsequent easy visualization.
Add an adequate volume of fluorescently-labeled EVs to an adherent recipient cell culture. Incubate for the desired period. During incubation, a sub-population of the labeled EVs superficially attach to the cell surface, while other EVs get internalized.
Add a different fluorescent dye to label the cell cytoplasm, helping distinguish internalized EVs from those adhered to the recipient cell. Place the cells under a confocal microscope and image at different focal planes along the z-axis, acquiring a series of optical sections across the sample thickness.
Compile this collection of optical sections - a z-stack - to form a high-resolution, three-dimensional sample image. Apply virtual rendering - a post-imaging process - to reconstruct the cell surfaces.
To differentiate superficially adhered from internalized EVs, render them as dots of different hues. Calculate the cell volume and number of EVs internalized by them, quantifying EV uptake per cell.

extracellular-vesicle-uptake-assay-method-to-visualize-quantify

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

In this video, we demonstrate an assay to visualize and quantify the cellular uptake of extracellular vesicles or EVs. This assay utilizes 3D fluorescent imaging using a confocal microscope and allows for distinguishing internalized EVs from ones adhered to the cell surface.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21154/21154_Figure1.jpg" /> 
Figure 1: Schematic illustration of the EV isolation and on-chip labeling using a nano-filtration-based microfluidic device.&#160;(A) EVs isolation from CCM. (B) On-chip Immunofluorescent labeling of EVs. (C) Removal of unbound antibodies.
<img alt="Figure 2" src="/file...

Extracellular Vesicle Uptake Assay: A Method to Visualize and Quantify Cellular Uptake of Extracellular Vesicles Using 3D Fluorescence Imaging Technique

In This Article

Overview

Protocol

Representative Results