Exosome Isolation and Purification Kit (From Milk)

1. Experiment Preparation

Pre-cool the centrifuge at 4°C for 10 min before use.

2. Sampling: For frozen samples, thaw them in a 25°C water bath and then place on ice for use. For fresh samples, directly place on ice for use.

Note: It is recommended that ≥ 25 mL of sample be used for a single extraction.

3. Centrifugation to Remove Impurities

Centrifuge at 4°C, 10,000 g (9,500 rpm) for 20 min. After centrifugation, the sample will separate into three layers: a top lipid layer, a middle whey layer, and a bottom protein precipitate. Transfer the middle whey layer to a 50 mL centrifuge tube.

Note: a. When collecting the middle layer, the lipid layer can be gently pierced with a pipette tip to allow slow pouring or pipetting of the middle layer.

b. It is normal for the collected whey to contain small amounts of the lipid layer and protein precipitate, and this generally does not affect downstream applications.

c. If the top layer appears “loose” or “easily detached” and the bottom precipitate is substantial, the centrifugation step can be repeated, each time collecting the middle layer.

d. The above centrifugation steps are based on a large centrifuge (effective radius ~ 10 cm) and ≥ 15 mL centrifuge tubes. The same applies below.

1. Add Solution A the collected whey according to the ratio in Table, immediately seal the centrifuge tube and invert it to mix thoroughly until the mixture appears “semi-transparent”.

2. Add Solution B, invert to mix and incubate at 4°C for 10 min.

Note: After incubation, gently shake the centrifuge tube. The solid should appear “tofu-like,” and the liquid should be “transparent.” If the solid does not form a “tofu-like” consistency or the sample remains “milky,” additional buffer B can be added until the liquid becomes “transparent”.

3. Centrifuge the clarified whey at 4°C, 10,000 g (9,500 rpm) for 120 min, and collect the supernatant.

4. Transfer the collected supernatant to a centrifugal filter column (50 mL) and centrifuge at 4°C, 5,200 g (3,000 rpm) for 2 min.

Note: a. If filtration cannot be completed in a single spin, perform multiple rounds of centrifugation.

b. The centrifugal filter columns are single-use consumables and are not recommended for reuse.

5. Add Solution C to the filtered supernatant and mix by gentle inversion.

1. Add Solution D to the sample mentioned above, immediately seal the centrifuge tube, and vortex for 1 min to mix thoroughly. Incubate at 4°C for at least 1 h.

Note: Extending the incubation time may increase exosome yield, but do not exceed 24 h.

2. Centrifuge at 4°C, 10,000 g (9,500 rpm) for 60 min, and collect the pellet, which is rich in exosome particles. Carefully remove the supernatant completely.

3. Centrifuge at 4°C, 10,000 g (9,500 rpm) for 2 min, and collect the pellet.

4. Resuspend the exosome pellet in an appropriate amount of PBS (1×) to dissolve it thoroughly and transfer it to a 1.5 mL centrifuge tube.

Note: It is recommended to resuspend the exosome pellet in 160 μL PBS for every 20 mL of sample.

5. Centrifuge at 4°C, 12,000 g (12,400 rpm) for 2 min, and collect the supernatant. The supernatant contains the exosome particles.

Note: a. If a significant amount of pellet remains, repeat the centrifugation until no visible pellet is observed.

b. The exosome solution may appear slightly milky, which is normal.

c. The above centrifugation steps are based on a benchtop centrifuge (effective radius ~ 7 cm) and ≤ 2 mL centrifuge tubes.

Aliquot the purified exosomes and store at -80°C for future use. Avoid repeated freeze-thaw cycles.