Exosomes are small vesicles (approximately 30-150 nm in diameter) secreted by living cells, characterized by a typical lipid bilayer structure. They are found in cell culture supernatants, serum, plasma, saliva, urine, amniotic fluid, and other biological fluids. Exosomes carry vital information including various proteins, lipids, DNA, and RNA. They not only play a crucial role in intercellular material and information transfer but also hold great promise as early diagnostic biomarkers for various diseases.

This kit employs a unique isolation technology with optimized components, specifically designed for the extraction of exosomes from emulsions. It allows for the rapid and efficient isolation of exosome particles, which are suitable for electron microscopy analysis, NTA particle size analysis, nucleic acid analysis, protein analysis, as well as cellular and animal experiments.

Product Information

Components

Materials Required but Not Supplied: 50 mL centrifuge tubes, 1.5 mL centrifuge tubes, 1×PBS buffer (sterile).

Storage

Shipped at room temperature. Store upright at 2-8°C. Shelf life: 1 year.

Instructions

I. Sample Pre-processing

1. Sampling: For frozen samples, thaw them in a 25°C water bath after removing from the freezer. Once completely thawed, place the sample on ice. For fresh samples, place them on ice immediately after collection.

2. Initial Sample Volume: The recommended volume of emulsion for a single extraction is 25-50 mL.

3. Lipid Removal: Transfer the sample to a centrifuge tube and centrifuge at 4°C, 10,000×g for 30 min to remove lipids and partial proteins from the sample. (Note: After centrifugation, the sample separates into three layers: the upper layer is the lipid layer, the lower layer is the protein precipitate, and the middle layer is the whey. After centrifugation, the upper layer should appear "dense, stable, and not easily dislodged". If the upper layer is "soft, easily dislodged" and there is a large amount of lower precipitate, repeat this step, collecting the middle layer liquid each time.)

4. Whey Transfer: Transfer the lipid-removed whey (the middle layer liquid) to a new 50 mL centrifuge tube. (Note: You can pierce the upper lipid layer with a pipette tip and pour slowly, or transfer it using a pipette. It is normal for the transferred whey to contain a small amount of lipids and precipitate, which will not affect subsequent experiments.)

II. Removal of Contaminating Proteins

1. Whey Clarification: Add Solution A to the whey and invert the centrifuge tube to mix. Then add Solution B, invert to mix, and let it stand at 2-8°C for 5 min. (Note: After standing, gently shake the centrifuge tube. It should present a "tofu-curd-like" solid, with the liquid portion appearing "transparent". If the "tofu-curd-like" appearance is not observed, add an appropriate amount of Solution B until this state is achieved after mixing and gently shaking the tube wall.)

[Note]: The specific volumes of Reagents A and B can be converted proportionally based on the table above.

2. Clarified Whey Centrifugation: Centrifuge the clarified whey at 4°C, 10,000×g for 20 min, and collect the supernatant. (Note: If white particles are visible in the supernatant, it is recommended to repeat this step to avoid difficulties in the next filtration step.)

3. Supernatant Filtration: Transfer the collected supernatant to a 50 mL centrifugal filter column and centrifuge at 4°C, 3,000×g for 10 min. (Note: If not fully filtered, repeat the centrifugation until all the upper liquid has passed through.)

4. Buffer System Equilibration: Transfer the filtered supernatant to a new 50 mL centrifuge tube, add Solution C, and invert to mix. (Note: The added volume of Solution C should be kept consistent with the volume of Solution B.)

III. Exosome Extraction

1. Add Exosome Extraction Solution: Add Solution D to the supernatant containing Solution C. The specific added volume is as follows (other volumes can be calculated proportionally based on the table):

2. Solution Mixing: After adding Solution D, tighten the centrifuge tube cap and mix thoroughly using a vortex mixer for 1 min. Then place it at 4°C and let it stand for more than 4 hours. (Note: Increasing the standing time can improve the exosome yield, but it should not exceed 24 hours.)

3. Exosome Precipitation: Take out the 50 mL centrifuge tube containing the mixed solution and centrifuge at 4°C, 10,000×g for 30 min. Discard the supernatant; the pellet is rich in exosome particles. (Note: Aspirate the supernatant as completely as possible.)

4. Secondary Centrifugation: Centrifuge the tube containing the pellet again at 4°C, 3,000×g for 5 min, and discard the supernatant. (Note: Aspirate the supernatant as completely as possible.)

5. Exosome Resuspension: Take an appropriate amount of 1×PBS and pipette up and down evenly to resuspend the centrifugal pellet. After it is dissolved, transfer the resuspension to a new 1.5 mL centrifuge tube. (It is recommended to use about 200 μL of 1×PBS to resuspend for every 20 mL of whey.)

6. Harvesting Exosome Particles: Centrifuge the 1.5 mL centrifuge tube containing the resuspension at 4°C, 10,000×g for 2 min. Retain the supernatant, which is rich in exosome particles. (Note: If there is a large amount of precipitate, centrifuge multiple times at 10,000×g for 2 min until no obvious precipitate remains, collecting the supernatant each time. The exosome solution may have a faint milky white color, which is a normal phenomenon.)

[Note]: Sterile treatment of exosomes: If sterile treatment is required, the exosomes can be filtered through a 0.45 μm filter before use.

IV. Exosome Purification

1. Exosome Purification: Transfer the harvested crude exosome particles into the upper chamber of 41209-E. Centrifuge at 4°C, 3,000×g for 10 min. After centrifugation, collect the liquid at the bottom of the column tube; this liquid is the purified exosome particles. (Note: The centrifugal filter column cannot be reused.)

2. Exosome Storage: Aliquot the purified exosomes in appropriate volumes and store them frozen in a -80°C freezer for subsequent experiments.

Notes

1. If further purification of exosomes is desired, affinity purification using magnetic beads coated with corresponding antibodies is recommended.

2. This kit is suitable for raw milk samples such as cow milk, goat milk, mare milk, and camel milk. It is not recommended for use with milk powder samples.

3. This product is specifically designed for milk/emulsion samples and is not suitable for the extraction of exosomes from serum/blood or cell culture supernatants. For exosome isolation from cell culture supernatants, please use the Cell Culture Supernatant Exosome Rapid Extraction Kit (Cat. No. 41205ES).

4. For your safety and health, please wear a lab coat and disposable gloves during operation.

5. This product is for scientific research use only!

Documents:

Safety Data Sheet

41209_MSDS_HB260525

Manuals:

41209_Manual_HB20260515

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