Exosomes refer to small membrane vesicles (30-150 nm) containing complex RNA and proteins, specifically disc-shaped vesicles with a diameter of 40-100 nm. Exosomes can be secreted by various cells under normal and pathological conditions. These exosomes play a key role in cell communication and genetic expression by transporting key proteins and genetic material. Exosomes naturally exist in body fluids, including blood, saliva, urine, cerebrospinal fluid and breast milk. Centrifugation is usually used to separate exosomes.
1. Experimental principle
Using the physical principle of molecular size and gravity and the precipitation principle of polymers, centrifugation is the most commonly used method to separate exosomes. This method can be extracted in large quantities, is low-cost, simple to operate, and takes a short time. Isolated exosomes can be used as therapeutic targets, drug or gene carriers, and as disease markers. They also play an important role in the transfer of substances and information between cells.
2. Experimental steps (different samples have different separation steps, taking urine as an example)
2.1 Thaw the frozen sample in a 25 ℃ water bath and place the completely thawed sample on ice.
2.2 Transfer 25 mL of urine to a centrifuge tube and centrifuge at 4 ℃, 3000 g for 10 min to remove cell debris; if there is a lot of precipitation, repeat the centrifugation several times until there is no obvious precipitation, and take the centrifuged supernatant.
2.3 Transfer the centrifuged supernatant to a 50 ml centrifugal filter column; centrifuge at 4 ℃, 3000 g for 10 min, and take the liquid in the lower chamber of the filter column; if there is residual liquid in the upper chamber, repeat this step to obtain more sample volume.
2.4 Add Exosome Concentration Solution (ECS reagent) to the supernatant after centrifugation and 5 ml ECS to 25 mL of urine.
2.5 Vortex and mix for 1 min and place at 4 ℃ for 2 h.
2.6 Take out the centrifuge tube containing the mixed solution and centrifuge at 4 ℃, 10000 g for 1 h, mark the precipitate position with a marker pen, discard the supernatant, the precipitate is rich in exosome particles, let it stand for 1 min, and then discard the supernatant again.
2.7 Take Exosome Solution Buffer (ESB reagent) and blow the centrifuged precipitate evenly, add 0.5 mL ESB to 25 mL urine, wait for it to dissolve, and transfer the resuspended liquid to a new 1.5 mL centrifuge tube.
2.8 Centrifuge at 4 ℃, 12000 g for 5 min, and retain the supernatant, which is rich in exosome particles. Store in aliquots at -80 ℃ for subsequent experiments.
3. Example of results
Schematic diagram (filtration method): First, remove cells and cell debris, then filter out free proteins and concentrate the sample. Finally, remove extracellular vesicles larger than 100 nm to obtain exosomes.
References
[1] Xu D, Tahara H. The role of exosomes and microRNAs in senescence and aging. Adv. Drug Deliv. Rev. 2013;65:368–375.
[2] Lobb, R. J. et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles. 4 27031 (2015).
[3] Vergauwen, G. et al. Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Scientific Reports. 7 (1), 2704(2017).
[4] Welton, J. L., Webber, J. P., Botos, L. A., Jones, M., & Clayton, A. Ready-made chromatography columns for extracellular vesicle isolationfrom plasma. J Extracell Vesicles. 427269 (2015).