Evaluation of exosome separation from human serum by frit-inlet asymmetrical flow field-flow fractionation and multiangle light scattering
»
Evaluation of exosome separation from human serum by frit-inlet asymmetrical flow field-flow fractionation and multiangle light scattering
Exosomes is 50- 150-nm-diameter extracellular vesicles secreted by all mammalian cell except mature red blood cells and contribute to a variety of physiological and pathological functions in the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies in the trial and raise the question of how to compare the results obtained using different approaches. Questions have also been raised about the purity of the preparations relating to the size and type of lipoprotein vesicles and presence.
Thus, researchers often find it challenging to identify exosome isolation protocol is optimal for their experimental needs. Our laboratory has compared ultracentrifugation and commercial precipitation- and column-based exosome isolation kit for exosome preparations. Here, we present a protocol for exosome isolation using the two most commonly used methods, ultracentrifugation and precipitation, followed by downstream analysis. We used NanoSight nanoparticle tracking analysis and flow cytometry (Cytek®) to determine the concentration and size of the exosome.
Basic Protocol 1: Pre-analytic fluid collection and processing base of Protocol 2: Exosome isolation by ultracentrifugation Alternative Protocol 1: Exosome isolation by precipitation basis of Protocol 3: Analysis of exosomes by NanoSight analysis tracking of nanoparticles Alternative Protocol 2: Analysis of exosomes by flow cytometry and imaging flow Basic cytometry Protocol 4: Downstream analysis of exosomes using high-performance liquid chromatography Basic Protocol 5: Downstream analysis of exosome proteome using nano-flow liquid chromatography-mass spectrometry.
Evaluation of exosome separation from human serum by frit-inlet asymmetrical flow field-flow fractionation and multiangle light scattering
Small insulation extracellular vesicles From Human Serum Using a combination of ultracentrifugation With Polymer-Based Deposition.
Exosome separation of lipoproteins validated by western blotting with several markers of exosomes and lipoproteins, followed by proteomic analysis using a nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. UC requires a relatively large amount of serum sample (at least 2 mL) but is more efficient in eliminating lipoproteins. UF method with centrifugal concentrators (300 kDa) were found to be more effective in taking serum exosomes with a low volume (50 uL). Overall, this study demonstrates the application of programming field FIAF4 for isolation / purification of exosomes from serum proteins and lipoproteins.
Methods to reproducibly isolate and enrich the small extracellular vesicles (EV) of blood is very important for clinical utilization of small EVs in cancer patients. We combine ultracentrifugation (UC) with rainfall based polymer (ExoQuick [EQ] or Total Exosome Isolation [TEI] kit) to isolate the small EVs (diameter, 30-150 nm) from the serum of breast cancer patients.
Rat Complete SeraMir Exosome RNA Amplification and Profiling Kit for Media and Urine (contains cat# RA800TC-1 with 50ml ExoQuick-TC, RA805A-1 and RA812A-1 components)
Description: This product includes 40 ml of Solution #5 for large-scale preparative extraction of membrane proteins (up to 130 mg) from the cell membranes.
Description: EXOSC5 Human Recombinant produced in E. coli is a single polypeptide chain containing 256 amino acids (1-235) and having a molecular mass of 27.5kDa.;EXOSC5 is fused to a 21 amino acid His-tag at N-terminus & purified by proprietary chromatographic techniques.
We compared the performance of four cycles of UC (UC4x) with those from two cycles of UC followed by enrichment using EQ (UC2x → EQ) or TEI (UC2x → TEI) kit. The average concentration of smaller EVs isolated from 1 mL of serum using UC2x → EQ (139.0 ± 29.1 mg) and UC2x → TEI (140.4 ± 5.0 mg) was not different from those obtained by using UC4x (141, 8 ± 26.9 mg).
