Abstract
Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids. NBI exploits nickel cations to capture negatively charged vesicles. Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution. Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them. We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gram-negative bacteria.
Highlights
[Background] Extracellular vesicles (EVs) are heterogeneous membranous structures that cells release in the extracellular fluids
The most widely used method for EV isolation is the differential ultracentrifugation, in which large EVs of about 200-1,000 nm sediment at low speed (10,000 x g), while small EVs of about 50-200 nm pellet at high speed (100,000 x g), as described by Théry et al, 2006. This size-based classification cannot be exploited to infer the intracellular pathways of their biogenesis, as ectosomes generated by the outward budding of the plasma membrane can overlap in size with exosomes derived from the multivesicular bodies (MVBs)
We describe the procedure of nickel-based isolation (NBI) we recently developed as a low-cost and convenient method for isolating EVs (Notarangelo et al, 2019)
Summary
Phosphate-buffered saline (PBS 1x, pH 7.4) (Thermo Fisher®, catalog number: 10010023) 9. NaCl powder (Sigma®, catalog number: 450006) 11. EDTA [0.5] M (Thermo Fisher®, UltraPure pH 8.0, catalog number: 15575020) 12. Citric acid (Sigma®, catalog number: 251275) 13. TRIzolTM Reagent (Thermo Fisher®, catalog number: 15596026) 14. Isopropanol Molecular Biology Reagent (Sigma®, catalog number: I9516) 17. UltraPureTM DNase/RNase-Free Distilled Water (Thermo Fisher®, catalog number: 10977035) 19. RNA grade (Thermo Fisher®, catalog number: R0551) 20. 3. Eppendorf® Centrifuge 5702, non-refrigerated, with Swing-bucket Rotor (A-4-38) included adapters for 15/50 ml conical tubes (Eppendorf, catalog number: 5702 000.32) (used in paragraphs A-B-C of the procedure). 4. Eppendorf® Thermomixer Comfort (Eppendorf, catalog number: 5355.000.011) 5. Eppendorf® Centrifuge 5415 R (Eppendorf, model: Centrifuge 5415 R, catalog number: 22 62 140-8), refrigerated, with Rotor (Eppendorf, catalog number: F-45-24-11) (used in Procedure D) 7. 2100 Bioanalyzer Instrument (Agilent Technologies, catalog number: G2939BA) 8. -80 °C freezer
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