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

Protocolo

1. Magnetic Bead-based Exosome Extraction

1. Pipette a well-mixed solution of 5 µL of Streptavidin-coated magnetic microparticles into 495 µL of phosphate-buffered saline (PBS) buffer in a microcentrifuge tube to resuspend the beads. Wash and resuspend the beads with 500 µL of PBS three times using a magnetic rack. The rack is an array of magnets on the side of a housing unit that can hold the sample microcentrifuge tubes.
   1. For each wash, first, let the tubes sit on the rack for 1 min and then use a pipette tip to carefully remove the supernatant buffer without disturbing the beads.
   2. Place the tubes on a regular rack without magnets at the side. Add 500 µL of PBS into the tubes and use the pipette to mix the solution and beads together. Then, put the tubes back on the magnetic rack to again separate the beads from the solution.
   3. Perform this removal of the buffer via magnetization and resuspension in PBS a total of three times. This performs an initial wash of the magnetic particles.
2. Resuspend the beads into 490 µL of PBS buffer, with the tube placed on the non-magnetized portion of the magnetic rack. Pipette 5 µL of biotinylated mouse anti-human CD63 antibody at 1.0 mg/mL stock concentration into the mixture of beads. Use the pipette to mix the beads and antibody in solution.
3. Place the microcentrifuge tubes with a bead and biotinylated antibody mixture on a sample rotator. Set the rotator parameters for the sample rotator for reciprocal rotation at 90° tilting for 5 s and vibrating at 5° for 1 s. Rotate the sample-bead mixture tubes at these parameters for 30 min at RT.
4. Remove unbound antibody after conjugation.
   1. Following 30 min of rotation at RT, place the tubes back in the magnetic rack for 5 min.
   2. Perform three washes of beads by removing the liquid phase using a micropipette and wash with 500 µL of PBS. After the triple wash, resuspend the beads in 490 µL of casein-PBS and place them on the unmagnetized portion of the rack.
5. Exosome extraction using antibody-coated beads.
   1. Label each tube with the targeted sample ID. Pipette a 10 µL sample of serum or saliva into the microcentrifuge tube. Use the pipette to mix the sample and magnetic beads by pipetting several times.
   2. Place the tubes with sample and anti-human CD63 antibody beads on the rotator and rotate for 2 h at RT. Use the same rotator parameters as described in step 1.3.
   3. Following 2 h of sample rotating, perform a triple wash by magnetizing to separate beads from solution, removing liquid phase with a micropipette, and resuspending beads in 500 µL of Tris-HCl buffer. The resultant beads are now bound to the exosomes and are ready for the electrical field release and measurement.

Materiales

Name	Company	Catalog Number	Comments
Biotinylated anti-human CD63 Antibody	Ancell, USA	215-030
Dynabeads MyOne Streptavidin T1	Invitrogen, USA	65601
Neodynium Magnetics (1/10" dia. x 1/32" thick)	K&J Magnetics, USA	DH101
Ultrapure Distilled Water	Life Technologies, USA	10977-023
Anti-Fluorescein-POD, Fab fragments	Roche, Germany	11426346910
Phosphate Buffered Saline Solution	Life Technologies, USA	10010023
Casein/PBS	Fisher Scientific, USA	37532