Magnetic Bead-based Exosome Extraction: A Technique to Isolate Specific Exosomes from Biofluids

Exosomes are extracellular vesicular structures secreted by cells into biofluids. To capture specific exosomes from biofluids, begin by taking streptavidin-coated magnetic beads in a tube.

To this tube, add biotinylated anti-human antibodies. Biotin present on the antibodies has a high affinity for streptavidin present on the magnetic beads. This results in the conjugation of the antibodies with the magnetic beads.

Next, add a protein-rich blocking solution to the tube containing the antibody-labeled magnetic beads. The proteins in the blocking solution bind to the free sites on the magnetic beads and increase specificity of exosome binding.

Now, supplement the tube with a sample biofluid rich in exosomes. Incubate the tube with agitation. The exosomes in the biofluid display specific surface receptors, which help them bind to the antibodies attached to the surface of magnetic beads.

Next, place the tube in a magnetic field. Under the influence of magnetic field, the exosome-bound magnetic beads attach to the surface of the tube. Then, using a micropipette, aspirate the remaining biofluid from the tube.

Finally, remove the tube from the magnetic field and add a suitable buffer to elute the exosome-bound beads. These exosome-bound beads can be used for further downstream analysis.

Resuspend a stock solution containing 10 micrograms per milliliter of streptavidin-coated magnetic microparticles, and add 5 microliters of the solution to a microcentrifuge tube containing 495 microliters of PBS. To wash the beads, first, set the tube on a magnetic rack for 1 minute and carefully remove the buffer without disturbing the beads.

Then, transfer the tube to a non-magnetic rack. Add 500 microliters of PBS and pipette to wash the beads. Do this a total of three times. After the final wash, resuspend the beads in 490 microliters of PBS and then add 5 microliters of biotinylated mouse anti-human CD63 antibody from a 1 milligram per milliliter stock solution.

Mix the solution by pipetting. Program a sample rotator for reciprocal rotation at 90 degrees for 5 seconds and vibration at 5 degrees for 1 second. Place the tubes in the rotator and run the program for 30 minutes at room temperature.

Next, transfer the tubes to the magnetic rack for 5 minutes. Wash the beads three times with 500 microliters of PBS and then resuspend them in 490 microliters of PBS containing casein. Label a tube with the sample identification and then add 10 microliters of the sample.

Pipette several times to mix the sample with the antibody-conjugated beads. Next, transfer the tubes to the sample rotator and run the same program for 2 hours at room temperature. Then, wash the beads three times with 500 microlitres of a 1M Tris-Hydrochloride buffer using the magnetized rack. The exosomes are now bound to the magnetized beads.