This protocol provides a robust methodology for analyzing EVs at a cellular level, and a practical approach for efficient EV uptake and EV tracking analysis. This method of EV labeling eliminates co-precipitation of EVs and dye, thus enhancing the EV signals. EV uptake is measured by volumetric analysis to distinguish the internalized EVs from the superficial EVs.
EVs are active cargo, thus, their uptake allows the transfer of biochemical molecules. For research on EV-based drug delivery and cancer therapy, this technique can be an assessment tool. EV uptake plays a role in intercellular communication and is investigated in various research fields, such as cancer biology, neuroscience, and drug delivery.
This protocol potentially facilitates efficient EV uptake assay. For isolation of extracellular vesicles, or EVs, inject one milliliter of pre-processed cell culture media into the sample chamber of the nanofiltration-based microfluidic device. To operate the device, spin the device in the benchtop spinning machine at 3000 RPM for 10 minutes.
After spinning, remove the fluid from the waste chamber by pipetting or aspirating, then repeat this procedure twice for processing an additional two milliliters of the cell culture media. Next, to wash the isolated EVs, inject one milliliter of PBS into the sample chamber and spin the device in the benchtop spinning machine. For immunofluorescent labeling of the EVs, inject one microgram per milliliter of the EV-specific antibody into the elution hole of the device containing 100 microliters of isolated EVs.
Then incubate for one hour in the dark on a plate shaker to ensure the even distribution of the antibody across the sample. Next, attach an adhesive tape to the elution hole. Inject one milliliter of PBS into the sample chamber to wash out residual antibodies, then spin the device until the sample chamber is empty.
Repeat the wash by injecting another milliliter of PBS into the sample chamber, and spinning the device as demonstrated previously. After removing the residual fluid from the chamber, pipette the fluorescently-labeled EVs from the membrane chamber into an amber tube or micro tube. Protect the tube from light until use.
Seed four times 10 to the fourth PC3 cells into a 35-milliliter dish with one 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 one microgram per milliliter of CMTMR, and incubate in optimal cell culture conditions. For live cell imaging, place the prepared cells in the onstage 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 Build 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. EVs isolated from PC3 cell culture media using a nanofiltration-based microfluidic device were labeled with a fluoro-4-conjugated EV-specific antibody, and visualized using 3D confocal microscopy.
The internalized EVs were visualized as puncta and an individual EV.Post-processing of these images allows the visualization and quantification of EV internalization into the cells. The EV uptake assay results indicate that the level of EV uptake depends on the length of the incubation period. The number of internalized EVs can be normalized to the recipient cell volume to determine the actual rate of EV uptake for the specified cell.
This procedure allows for the systematic exclusion of non-internalized EVs to precisely measure the number of internalized EVs. The size distribution of internalized EV dots is shown here. So the key steps are the utilization of the microfluidic device for EV labeling, the minimization of background fluorescence when visualizing fluorescent EVs, and then the post-imaging analysis to determine internalized versus superficial EVs.
So intracellular EV tracking can be performed in real time on a 3D plane, and additionally, EV trafficking analysis on spatial/temporal resolutions and the co-localization of EVs with subcellular organelles can be achieved. So we believe this technique provides an easier, more efficient way to investigate EVs within the intra-and extracellular matrices for researchers within the cellular communication field for EVs.
