Serum/Plasma Exosome Isolation and Purification Kit (Magnetic Beads)

Sample Pretreatment

1. Blood Sample Collection

Collect serum or plasma samples according to experimental requirements and keep the samples on ice until use.

2. Sample Dilution

Transfer 600 μL of serum/plasma sample into a 1.5 mL centrifuge tube. Add an equal volume (600 μL) of Dilution Buffer and gently invert to mix thoroughly. Avoid vigorous shaking during operation to minimize potential damage to exosome integrity.

Magnetic Bead Pretreatment

Thoroughly resuspend the magnetic beads. Transfer 150 μL of magnetic beads into a 2 mL EP tube and add 1.2 mL of Magnetic Bead Wash Buffer. Mix gently. Place the tube on a magnetic rack (MCE Cat. No.: HY-K0200A) for 1-2 min for magnetic separation, or centrifuge at 3,000 g (6,200 rpm) for 5 min at 4°C. Discard the supernatant. Add Magnetic Bead Wash Buffer and repeat the washing step once.

Note: The above centrifugation conditions are based on a small centrifuge with an effective radius of approximately 7 cm and centrifuge tubes with a capacity of ≤ 2 mL. For larger sample volumes or centrifugation systems, it is recommended to divide the sample into multiple tubes. The same applies to the centrifugation steps below.

Exosome Extraction

1. Preparation of the Exosome Extraction System

Add the pretreated serum/plasma sample (1.2 mL) to the pretreated magnetic beads described above and mix gently.

Note: The ratio of magnetic beads to sample has been optimized. For larger sample volumes, the amounts of all components may be increased proportionally.

2. Magnetic Bead-Sample Incubation

Seal the centrifuge tube with sealing film and place it on a rotary mixer. Incubate with rotation at 4°C for 30 min.

3. Complex Collection

After incubation, place the centrifuge tube on a magnetic rack for 1-2 min for magnetic separation, or centrifuge at 3,000 g (6,200 rpm) for 5 min at 4°C. Discard the supernatant.

4. Magnetic Bead Washing

Slowly add 120 μL of Magnetic Bead Wash Buffer along the inner wall of the centrifuge tube without mixing. Immediately place the tube on a magnetic rack for 1-2 min for magnetic separation, or centrifuge at 3,000 g (6,200 rpm) for 5 min at 4°C. Discard the supernatant.

Note: Do not mix or pipette after adding the wash buffer in this step. Excessive washing may reduce exosome recovery.

5. Exosome Elution

Add 120 μL of Elution Buffer to the centrifuge tube and vortex for 30 s to fully elute the exosomes.

6. Exosome Collection

Centrifuge the tube at 7,000 g (9,500 rpm) for 2 min at 4°C, then place it on a magnetic rack for 1-2 min. Transfer the supernatant to a new centrifuge tube. The collected supernatant is the crude exosome preparation.

7. Repeated Elution

Repeat the exosome elution and collection procedures described above (Steps 5-6), and combine the two exosome eluates.

Exosome Purification and Storage

1. Exosome Purification

Transfer the collected crude exosome preparation to the upper chamber of the exosome purification column and centrifuge at 3,000 g (6,200 rpm) for 10 min at 4°C. Collect the liquid at the bottom of the purification column tube as the purified exosome preparation.

2. Exosome Storage

Aliquot the purified exosomes and store at -80°C until use. Avoid repeated freeze-thaw cycles to preserve exosome integrity and associated biological activity.

Note: Freshly prepared purified exosomes are recommended for subsequent experiments and analyses to obtain optimal results.