This article describes a method for capturing and analyzing extracellular vesicles (EVs) using magnetic nanoparticles (MNPs) conjugated with fluorescent antibodies. The process involves incubating MNPs with EVs, blocking non-specific binding, and analyzing the resulting complexes via flow cytometry.
Begin with a tube carrying cell-derived membrane-bound particles termed extracellular vesicles, or EVs, that exhibit unique antigenic compositions.
Introduce magnetic nanoparticles, MNPs, containing capture antibodies conjugated with fluorescent-labeled Fab fragments.
Incubate to facilitate the interaction between the MNPs with specific antigens of EVs, forming MNP-EV complexes.
Add immunoglobulin-G to block Fc binding sites, then introduce fluorescently labeled detection antibodies to stain other antigens on the captured particles.
Load this mixture onto a pre-washed magnetic column placed in a magnetic separator.
The magnetic field retains MNP-captured particles within the column. Exchange the buffer to remove unbound antibodies.
Retrieve the column from the magnetic separator, elute MNP-captured particles with a buffer, and fix them to preserve their structural integrity.
Analyze the immunocaptured particles by flow cytometry; distinct fluorescence signals from stained antigens correlate with unique antigenic characteristics of EVs.
Begin by combining 60 microliters of MNPs per condition and 5 microliters of labeled Fab fragments specific for the isotype of the capture antibody of interest in a 1.5-milliliter microcentrifuge tube. Incubate the solution at room temperature for 30 minutes with continuous mixing. Then, pre-wet a 100k concentrator with 300 microliters of PBS, and spin the concentrator in a microcentrifuge.
At the end of centrifugation, purify the labeled complexes on a 100k column, followed by another centrifugation, resuspending the pellet in 200 microliters of fresh PBS. To capture the particles of interest, incubate the antibody-labeled MNPs with the particle of interest for the appropriate incubation period and temperature.
Next, block any non-specific FC binding with the appropriate species of IgG antibody, gently vortex, and incubate the particle solution for 3 to 5 minutes at room temperature. Then, add the manufacturer-recommended concentration of each detection antibody to the particles or isotype control antibodies to the isotype control sample for an additional 20 minutes incubation at room temperature.
To separate the MNP-captured particles from the unbound antibody, first, place a magnetic separation column in a separator magnet and pre-wet the column with 500 microliters of wash buffer. Allow the wash buffer to flow through the column. Then, add the MNP particle complexes to the column, collecting the flowthrough in a waste container.
When all of the complex solution has passed through the column, wash the column three times with 500 microliters of wash buffer. Then, transfer the column to a 12-by-75 millimeter tube. After three minutes, add 200 microliters of PBS, allowing the beads to elute by gravity.
When all of the PBS has passed through the column, add 200 microliters of fresh PBS to the tube, and fix the particles with 200 microliters of 4% paraformaldehyde. Next, load 0.22-micron-filtered PBS onto a flow cytometer and adjust the PMT voltage on the cytometer until the filtered PBS shows zero to very few events. Then, run the MNP-captured particles using a low flow with a 0.04-micron in-line filter for sheath fluid placed in series behind the standard 0.2-micron sheath filter to further eliminate any false events.
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