Exosomes are tiny vesicles with a diameter of 30-200 nm, which can be released by many types of cells in the body and are widely distributed in body fluids such as saliva, plasma, milk, and urine. Exosomes can carry a variety of proteins, mRNA, and miRNA, and participate in processes such as cell communication, cell migration, angiogenesis, and tumor cell growth.
Ultracentrifugation is the gold standard method for isolating exosomes from biological fluids or cell supernatants. Using low-speed centrifugation and high-speed centrifugation alternately, vesicle particles of similar size can be separated, which is a classic technical method commonly used at present. The isolated exosomes have high purity and high yield, which can meet the needs of different subsequent experiments.
Creative Proteomics has ultra-high-speed centrifuges with high efficiency and guaranteed quality. The samples for ultracentrifugation exosome extraction service include cell culture supernatant, serum, plasma, urine, cerebrospinal fluid, etc. The obtained high-purity exosome particles can be used for subsequent exosome analysis such as NTA, TEM, and WB.
Figure: Schematic representation of Ultracentrifugation-based Exosome Isolation. Source (Pin Li et al., Theronostics, 2017)
Our Exosome Service
In addition, Creative Proteomics can provide a variety of exosome isolation and extraction solutions according to the characteristics of customer samples, such as filtration centrifugation, kit extraction, etc. Also, we provide customized exosome proteomics analysis to identify and quantify the proteins in the exosomes with the obtained high-purity exosome particles.
Service Process
1. Collection of culture medium (storage and transportation on dry ice).
2. Centrifuge at low speed to remove cells, debris and impurities.
3. Obtain the exosome pellet by ultracentrifugation.
4. Exosome purification.
5. BCA method was used to detect the concentration of exosomes.
6. Provide experiment report.
Exosome Extraction
Creative Proteomics uses ultracentrifugation to extract exosomes from cell culture supernatant, serum, plasma, urine, cerebrospinal fluid and other body fluids (ascites, amniotic fluid, milk and saliva, etc.) to obtain high-purity exosome particles.
Sample Preparation
Cell culture supernatant
Cell culture medium must use exosome-depleted serum (such as exosome depleted Fetal Bovine Serum) or serum-free medium to reduce background interference (bovine serum contains a large amount of bovine exosomes).
1) The cells are cultured for a certain period of time in a medium containing normal serum, and the cells grow to about 70%;
2) For adherent cells, remove the original medium, wash with PBS, and replace with a new medium containing exosome-free serum or serum-free medium; for suspension cells, collect cells at 300×g, 4°C, for 10 minutes , wash with PBS, use exosome-free medium or serum-free medium to suspend the cells and continue to culture;
3) Continue to culture the cells for 24-48 hours, and determine the time to collect the supernatant according to the growth rate of the cells;
4) Collect the cell supernatant, centrifuge at 300×g, 4°C for 10 minutes; carefully aspirate the supernatant, and avoid aspirating cells or cell debris;
5) Filter the supernatant at 0.22 µm to remove cell debris, apoptotic bodies and microvesicles;
6) Freeze the supernatant sample at -80°C.
Suggested sample volume: 20ml or more
Plasma
1) Take whole blood into an EDTA anticoagulant tube and mix gently;
2) Centrifuge at 2500×g for 15 minutes at 4°C, and take the supernatant;
3) Centrifuge again at 2500×g for 15 minutes, and take the supernatant as plasma;
4) Store plasma at -80°C.
Suggested sample volume: 2ml or more
Serum
1) Put 4ml of whole blood in a common blood collection tube (note, do not add anticoagulant to collect serum samples) and coagulate at 37°C for 20-30 min;
2) Centrifuge at 2000×g for 10 min, take the supernatant, and the light yellow transparent liquid is the serum;
3) Store the serum at -80°C.
Suggested sample volume: 2ml or more
Urine
1) Collect 50ml of urine for the first time in the morning in a sterile container;
2) Add protease inhibitor mixture (1.67 ml of100mM NaN3, 2.5ml PMSF (2 mg/ml in isopropyl alcohol), 50 µl Leupeptin (1 mg/ml in ddH2O));
3) Process immediately or store at -80°C.
Suggested sample volume: 20ml or more
Cerebrospinal fluid
1) Collect cerebrospinal fluid and place it on ice for 30 minutes;
2) Centrifuge at 2000×g for 10 minutes at 4°C to remove cells or cell fragments;
3) Take the supernatant and process it immediately or store it at -80°C.
Suggested sample volume: 10ml or more