Exosomes are small vesicles (30–120 nm) containing RNA and protein that are secreted by various types of cells in culture, and found in abundance in body fluids, such as blood, saliva, urine, and breast milk. Exosomes were reported to function as intercellular messengers, delivering their cargo of effector or signaling macromolecules between specific cells; however, their formation, the makeup of the cargo, and biological pathways in which they are involved remain unclear.
The biological & medical study of exosome function and trafficking requires the isolation of intact exosomes, but the current methods used are tedious and difficult. The EZBioscience Exosome Isolation Kit (from cell culture media) provides a simple and reliable method of concentrating intact exosomes from cell culture media samples. By tying up water molecules, the Exosome Isolation reagent forces less-soluble components (i.e. exosomes) out of solution, allowing them to be collected with a brief and relatively low-speed centrifugation.
After Isolation, the exosome can be used for further experiments, auch as Immuno-blotting and RNA purification, RNA sequencing, and RT-qPCR etc. .
Experimental Procedure at a Glance
Protocol
General Guidelines
a) To ensure that isolated exosomes originate from your cells of interest, culture the cells with exosome depleted fetal bovine serum ( FBS ) or serum-free culture medium, because normal FBS contains extremely high levels of exosomes that will contaminate the cell derived exosomes. If you cannot obtain exosome depleted FBS, certain cell lines can be grown for up to 12 hours in media without FBS.
b) The Exosome Precipitation Reagent (EPR) is not recommended for isolation of exosomes from other kinds of samples. If you are isolating intact exosomes from plasma, use the Total Exosome Isolation (from plasma) reagent.
Prepare Sample
1. Harvest 5~20 ml cell culture media ( or urine, broncho alveolar lavage fluid, cerebro-spinal fluid )..
2. Centrifuge the plasma sample at 3000 × g for 10 minutes at 4°C to remove cells and debris.
3. Transfer the supernatant containing the cell-free culture media to a new tube without disturbing the pellet.
Isolate Exosomes
1. Transfer the required volume of cell-free culture media to a new tube and add 0.25 volume of the Exosome Precipitation Reagent (EPR).
Culture Media | Reagent |
5 ml | 1.25 ml |
20 ml | 5 ml |
2. Mix the culture media/reagent mixture well either by vortexing or inversion the tube until the solution is homogenous. Incubate the sample at 2 ~ 8°C overnight.
3. After incubation, centrifuge the sample at 10000 × g for 30 min at 4°C.
4. Carefully aspirate the supernatant by pipetting and discard the supernatant. Exosomes are contained in the pellet at the bottom of the tube (not visible in most cases).
Resuspend Exosomes
1. Add 1× PBS or similar buffer to the pellet and vortex or pipette up and down to resuspend the exosomes.
Starting Culture MediaVolume | Resuspension Volume |
1 ml | 30 µl |
10 ml | 60 µl |
2. Once the pellet is resuspended, the exosomes are ready for downstream analysis or further purification through affinity methods.
Note: The purified exosome sample may be stored at 2 ~ 8°C for up to 7 days or can be stored below -80°C for long term storage. To minimize the risk of RNase contamination, we recommend proceeding directly with further downstream sample processing.
For detailed information, please check the product manual.
