Exosome Isolation and Purification Kit (From Tissue) 

Exosomes are small vesicles (30 - 150 nm) containing RNA and protein that are secreted by various types of cells in culture, and found in abundance in body fluids including blood, saliva, urine, and breast milk. Exosomes are thought to function as intercellular messengers, delivering their cargo of effector or signaling macromolecules between specific cells.

MCE Exosome Isolation and Purification Kit (From Tissue) provides a simple and effective method to isolate and purify intact exosomes from a variety of tissues (such as brain, heart, lung, liver, muscle, lymph nodes, thymus, embryo, tumor, etc.). The obtained exosomes are of high purity and activity, making them suitable for applications such as electron microscopy analysis, NTA analysis, WB, qPCR, cell biology studies, and animal experiments.

Solution A2: -20°C, 2 years.

Solution B2, Exosome Purification Filter 2 (EPF): RT, 2 years.

1. During sample preparation, if the sample concentration is too high or too low, appropriate dilution or concentration can be performed. Centrifugation can be repeated until no significant pellet is observed.

2. The products in this kit do not contain RNase or DNase. During the experiment, avoid introducing RNase and DNase contamination.

3. It is recommended to use freshly prepared samples and avoid repeated freeze-thaw cycles.

4. If you need to co-culture the obtained exosomes with cells later, you can filter them through a 0.22 μm filter membrane to remove bacteria.

5. This product is for R&D use only, not for drug, household, or other uses.

6. For your safety and health, please wear a lab coat and disposable gloves to operate.

1. Experiment Preparation

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

2) Place Solution A2 in a 4°C refrigerator to thaw, then take an appropriate amount of Solution A2 and keep it on ice for later use.

Note: It is recommended to aliquot the Solution A2 after the first thaw to avoid repeated freeze-thaw cycles.

2. Tissue Preprocessing: Cut the tissue into 1 mm³ - 3 mm³ pieces (the smaller the better) in sterilized containers (placed on ice), and transfer to a centrifuge tube. Add ≥ 10 times the volume of PBS (1×) and mix thoroughly. Centrifuge at 300 g (2,000 rpm) for 10 min and discard the supernatant.

Note: The above centrifugation steps are based on a small centrifuge (effective radius ~ 7 cm) and ≤ 2 mL centrifuge tubes. For larger systems, it is recommended to centrifuge in separate tubes, the same applies below.

3. Tissue Digestion: Add 1 mL of Buffer A2 for every 0.1 g of tissue. Mix the tissue fragments with Buffer A2 in a 2 mL centrifuge tube and incubate on a horizontal shaker at 37°C, 80 rpm for 20 min.

Note: Alternatively, incubation can be performed in a 37°C water bath for 30 min, and it is recommended to mix manually every 5 min.

4. Centrifugation to Remove Impurities

1) Centrifuge at 8,000 g (10,100 rpm) for 10 min, and collect the supernatant.

2) Centrifuge at 4°C, 12,000 g (12,400 rpm) for 10 min, and collect the supernatant.

1. For every 1 mL of the preprocessed sample, add 250 μL of Solution B2. Mix the supernatant obtained from the preprocessing step with Solution B2, immediately seal the centrifuge tube, and vortex for 1 min to mix thoroughly. Incubate statically at 4°C for at least 1 h.

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

b. Solution B2 should be stored at room temperature. If the temperature is too low, white crystals or precipitates may form and can be difficult to re-dissolve (requiring high-temperature water bath for re-dissolution). A small amount of precipitate generally does not affect exosome extraction efficiency.

2. Centrifuge at 4°C, 12,000 g (12,400 rpm) for 30 min, and collect the pellet, which is rich in exosome particles. Carefully remove the supernatant completely.

3. Centrifuge at 4°C, 12,000 g (12,400 rpm) for 2 min, and collect the pellet.

4. Resuspend the exosome pellet in an appropriate amount of PBS (1×) to dissolve it thoroughly.

Note: It is recommended to resuspend the exosome pellet in 200 μL PBS for every 0.1 g of tissue 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: If a large amount of pellet is observed, repeat the centrifugation until there is no visible pellet.

1. Exosome Purification

Transfer the collected crude exosomes to the exosome purification filter’s upper chamber, and centrifuge at 4°C, 3,000 g (6,200 rpm) for 10 min. Collect the liquid at the bottom of the filter tube, which contains the purified exosomes.

Note: a. If a significant amount of liquid remains in the filter column after centrifugation, it can be transferred to a new filter for repeated centrifugation to recover exosomes.

b. Under normal conditions, the exosome yield from 0.1 g of tissue (e.g., myocardial tissue) is 100 - 200 μg.

2. Exosome Storage

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

1. During sample preparation, if the sample concentration is too high or too low, appropriate dilution or concentration can be performed. Centrifugation can be repeated until no significant pellet is observed.

2. The products in this kit do not contain RNase or DNase. During the experiment, avoid introducing RNase and DNase contamination.

3. It is recommended to use freshly prepared samples and avoid repeated freeze-thaw cycles.

4. If you need to co-culture the obtained exosomes with cells later, you can filter them through a 0.22 μm filter membrane to remove bacteria.

5. This product is for R&D use only, not for drug, household, or other uses.

6. For your safety and health, please wear a lab coat and disposable gloves to operate.