Characterizing Extracellular Vesicles from Biological Fluids

Extracellular vesicles serve as disease biomarkers, reflecting physiological states. Our approach enables rich data extraction from tiny tear volumes, advancing EV research in biological fluids. Key experimental challenges include obtaining sufficient extracellular vesicle concentrations from small samples, minimizing sample loss during isolation, and optimizing analysis to prevent chip oversaturation or weak signal detection.

This protocol bypasses isolation steps, reducing processing time, and allows extracellular vesicles analysis from minor sample volumes, making it ideal for limited biological samples, like tears. Non-invasive biomarkers are crucial for early detection and management of corneal disorders. Extracellular vesicle biomarkers enable disease monitoring, personalized treatments, better patient compliance, and improved quality of life.

Our lab pioneered the analysis of extracellular vesicles in tear samples from KC subjects, identifying unique tEV signatures. This paves the way for future diagnostics and EV-focused therapeutic targets. To begin, set out the biological samples and a tetraspanin chip on the workspace.

Pipette 70 microliters of human biological samples that have been premixed with solution B of a human tetraspanin kit onto a chip. Place the film on a chip washing plate to prevent sample evaporation. After 16 hours, place the plate in the chip washer.

Lock it in place and close the lid. Press the CW-TETRA v0 option on the chip washer. While the chips are being washed, prepare 300 microliters of blocking solution per chip.

Add 0.6 microliters of each antibody per chip to the blocking solution. Vortex the blocking solution with the antibody cocktail to ensure thorough mixing. When the chip washing is complete, add 250 microliters of the blocking solution with the antibody cocktail on top of each chip.

Then close the lid and press Continue. When the chip washer chirps, remove the chip washing plate. Move the chips up the ramp.

Place the plate back into the chip washer, ensuring it locks securely into place. Close the lid and press Continue. To begin, turn on a nanoparticle analyzer.

Launch the scanning software program on the desktop connected to the nanoparticle analyzer. Select the chip folder to choose each tetraspanin chip and locate it in Chip Position on the chuck. Click on the three squares below Chip Position to turn on the fluorescence.

The squares will turn yellow, blue, and red. Press on the chuck lid and pull it to remove it. Then firmly secure the sample-loaded tetraspanin chips on the chuck.

Align the pegs of the chuck lid with the holes and press down. Slide the lid to lock it into place. Then place the chuck onto the stage and lock it in place.

Next, click on Scan Chips from the software interface, then click on OK.Once the stage fully moves into the machine, close the door. When the scan is complete, open the door to eject the stage and verify results. If the scans were successful, discard the chips.

Place the next set of chips on the chuck. Alternatively, return the empty chuck to the stage. Exit the software and turn off the machine when finished.

Optimal fluorescence levels of extracellular vesicles were observed in the CD63, CD81, and CD9 channels, while the MIgG control channel remained black, confirming the control specificity. CD81 was reduced for biological fluids. No fluorescence in CD63, CD81 and CD9 channels is indicative of an absence of extracellular vesicles.

Oversaturation of fluorescence was evident, making it difficult to discern meaningful data due to excessive labeling, particularly in CD63, CD81, and CD9 channels.