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

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.

Seed 4 x 10⁴ PC3 cells into a 35-milliliter dish with 1-milliliter media. Allow the cells to adhere overnight in optimal cell culture conditions. The following day, wash the adhered cells twice with exosome-depleted media.

After diluting the fluorescently-labeled EVs to the appropriate concentration using exosome-depleted media, add the diluted EVs to the adhered target cells, and incubate for the desired experimental time. After removing non-internalized EVs by washing the cells thrice with exosome-free media, label the cytoplasm of the adhered cells with 1 µg/mL of CMTMR, and incubate in optimal cell culture conditions.

For live-cell imaging, place the prepared cells in the on-stage incubator. After setting the imaging parameters based on control samples, determine the depth of the target cells, and the range of stacking size in the z-direction to acquire 3D confocal images. Then, set the image acquisition to multiple z-stacked images of both cell-specific dye and EV-specific dye simultaneously.

For image processing, utilize automatic image processing software.

To build virtual surfaces of the cells, click on 'Add New Surfaces.' To configure the virtual cell surfaces, click the '+ Add' button and select 'Quality' as the "Filter type." Threshold the appropriate value for the low limit by visual inspection, set the maximum value for the upper limit, and click the 'Finish' button.

Next, to build the virtual dots of the EVs, click the button 'Add New Spots.' Under "Algorithm Settings," select 'Different Spot Sizes' and 'Shortest Distance Calculation.' Then, click 'Next,' and select "Channel 1 - Alexa Fluor 488" as the "Source Channel." Enter the appropriate value for the 'Estimated XY Diameter' under "Spot Detection" and click 'Next.'

To configure the virtual EV dots, click the '+ Add' button and select 'Quality' as the 'Filter Type.' Set the "Lower Threshold" by a visual inspection and click 'Next.' For the "Spot Regions Type," select 'Absolute Intensity,' then click 'Next.'

To threshold the region of EV dots, enter the appropriate value for "Region Threshold" by visual inspection. Select "Diameter from" under "Region Volume" and click 'Finish.'

To split the grouped spots inside the built surface, click on the built 'Spots' and go into 'Filters.' Next, click the 'Add' button and select 'Shortest Distance to Surfaces Surfaces = Surface 1' as the "Filter Type."

After setting the lowest threshold for the low limit and the appropriate value for the upper limit, click the 'Duplicate section to the New Spots' button.

For automatic counting of EVs inside the cells, click the built 'Spots 1 selection,' go to 'Statistics,' and export the value from the 'Total Number of Spots.'

To obtain the number of cells, click the built 'Surfaces 1,' then, go to 'Statistics' and export the value of "Total Number of Surfaces" from 'Overall.' Finally, go to the 'Detailed' tab under 'Statistics' to export the volume.