Evaluation of exosome separation from human serum by frit-inlet asymmetrical flow field-flow fractionation and multiangle light scattering
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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.
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: ML239 is the best-in-class inhibitor of the breast cancer stem cells with an IC50= 1.16 µM. [1]ML239 was a selective inhibitor an IC50= 1.18 µM against HMLE_sh_Ecad.
Description: IC50: 140 nMML-354 is an indole-based, proteinase-activated receptor (PAR) 4 antagonist with good potency and reasonable selectivity versus PAR1. PARs are a family of four G-protein-coupled receptors GPCRs (PAR1-PAR4) with several unique attributes, which are activated by serine proteases.
Description: ML-356 is an inhibitor of the thioesterase domain of fatty acid synthase with IC50 value of 334 nM.Fatty acid synthase (FAS) is a multi-enzyme protein that catalyzes fatty acid synthesis, fatty acids are related to energy production and storage, cellular structure ,hormones biosynthesis
Description: ML-291 is an apoptosis inducer. Apoptosis, a process of programmed cell death, occurs in multi-cellular organisms. Biochemical events result in characteristic cell changes (morphology) and death.
Description: IC50: 221 nMML-193 is a GPR55 antagonist. GPR55 is a class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer.
Description: ML-097 (CID-2160985) is a pan activator of Ras-related GTPases [1]. The Ras superfamily of GTPases, which includes Arf, Rho, Ras and Rab GTPase subfamilies, regulate many cellular processes ranging from membrane trafficking to the control of cell proliferation.
Description: IC50: 32, 20, and 41 nM for TC-83, V3526, and Trinidad donkey strains, respectively.ML-336 is an inhibitor for VEEV-induced cytopathic effect.
Description: IC50: LYPLA1 (17 nM) and the related LYPLA2 (30 nM)ML-211 is a dual inhibitor of LYPLA1 and the related LYPLA2.Lysophospholipase 1 (LYPLA1), a protein palmitoyl thioesterase, is responsible for depalmitoylation of the oncogene HRas.
Description: ML-265 (TEPP-46) is a potent and selective PKM2 activator [1][2]. Pyruvate kinase catalyzes the final step in glycolysis and transfers the phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) to yield adenosine triphosphate (ATP) and pyruvate.
Description: HIF pathway activator (EC50 values are 1.23 and 1.4 ?M for HRE gene reporter assay and HIF-1? nuclear translocation assay respectively); acts via chelation of iron, independently of PHD.
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).
