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.

Imaging flow cytometry can be used for both exosomes size and surface markers immunophenotype in exosomes (ImageStream®). High performance liquid -Kromatografi followed by nano-flow liquid chromatography mass spectrometry (LCMS) of exosome fractions can be used to determine the presence of lipoproteins, the LCMS is able to provide a proteomic profile of exosome preparations. We found that the method of precipitation is six times faster and resulted in ~ 2.5-fold higher concentration of exosomes per milliliter compared with ultracentrifugation. Both methods produce extracellular vesicles in various sizes of exosomes, and both preparations, including apoproteins. © 2020 Wiley Periodicals LLC.

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
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.

ExoQuick Exosome RNA Column Purification kit

EQ808A-1 20 preps
EUR 482.4

Exosome precipitation from Plasma and Serum

EPStep-PS 5 ml
EUR 356.4

Thrombin Plasma prep for Exosome precipitation (500 ul at 500U/mL)

TMEXO-1 100 reactions
EUR 699.6

ExoQuick-LP for lipoprotein pre-clear & exosome isolation

EXOLP5A-1 5 reactions
EUR 639.6

ExoQuick PLUS Exosome Purification Kit - for serum and plasma

EQPL10A-1 10 reactions
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LV CryoOil 5 ml

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EUR 64
Description: LV CryoOil 5 ml

ICP-MS Multi-Element Solution 5

CLMS-5 125ML
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ExoQuick-TC PLUS Exosome Purification Kit - for tissue culture media

EQPL10TC-1 10 reactions
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Proteinase K, Recombinant, 20 mg/ml Solution, Molecular Grade

9211-5 each
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Dispase Solution, 1mg/ml

CA092-010 100ml
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Transferrin Solution, 10mg/ml

CA205-005 5x1ml
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cleaning solution 500 ml

B530 ea
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Kanamycin solution (50mg/ml)

MN800A-1 20 ml
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Gentamicin Solution, Gentamicin Solution, 50 mg/mL

CCM1123-010 10 mL
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ExoQuick Exosome Isolation and RNA Purification kit (for Serum and Plasma)

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ExoQuick Exosome Isolation and RNA Purification kit (for Tissue Culture Media)

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SeraMir Exosome RNA Purification kit (5ml ExoQuick and 20 exoRNA columns)

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Hematoxylin Solution (5%)

HSV250 250 ml
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Hematoxylin Solution (5%)

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Hematoxylin Solution (5%)

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Exosome Component 5 Protein

20-abx260873
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  • 1 mg
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ExoQuick TC 10ml

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CH006 1.0 ml
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CH007 5.0 ml
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Sodium Selenite Solution,100mg/ml

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B231 ea
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pepsine cleaning solution 1000 ml

B232 ea
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RNase A Solution (10mg/ml)

RB0474 1ml
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Sodium chloride 5 M, 1000 ml

12-9191-5 1000 ml
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ExoPure? 0.4 micron Immunobeads (Overall Exosome Isolation, cell media, 5 reactions)

M1030-5 each
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M1031-5 each
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DNA Precipitation Buffer

D106 55 ml
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Exosome Component 5 (EXOSC5) Antibody

20-abx112414
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Exosome Component 5 (EXOSC5) Antibody

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Exosome Component 5 (EXOSC5) Antibody

20-abx302857
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HSA Solution 5% (cGMP)

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SeraMir Exosome RNA Purification kit (10ml ExoQuick-TC and 10 exoRNA columns) 10 preps

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5-Bromo-5-nitro-1,3-dioxane (solution)

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Exosome Component 5 (EXOSC5) Antibody (HRP)

20-abx311411
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  • EUR 218.40
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  • 100 ug
  • 1 mg
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Exosome Component 5 (EXOSC5) Antibody (FITC)

20-abx311412
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  • 100 ug
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Exosome Component 5 (EXOSC5) Antibody (Biotin)

20-abx311413
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  • 100 ug
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Finegel Stacking Gel Solution(5%)

F1505-010 100ml
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5 x PEG/NaCl Solution

GR103043 100 mL
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ML 239

A8719-5 5 mg
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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.

ML-354

C3058-5 5 mg
EUR 174
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.

ML-356

C3354-5 5 mg
EUR 474
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

ML-264

C3474-5 5 mg
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Description: ML264, a potent and selective inhibitor of kruppel-like factor 5 (KLF5), inhibits the growth of colorectal cancer.

ML-031

C5718-5 5 mg
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Description: ML-031 is an agonist of S1P2 with EC50 value of 1 ?M [1].

ML-291

C5358-5 5 mg
EUR 201.6
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.

ML-193

C4531-5 5 mg
EUR 216
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.

ML-099

C4771-5 5 mg
EUR 219.6

ML-191

C4786-5 5 mg
EUR 219.6

ML-233

C4788-5 5 mg
EUR 219.6

ML-335

C4792-5 5 mg
EUR 189.6

ML-030

C4805-5 5 mg
EUR 219.6

ML-098

C4807-5 5 mg
EUR 219.6

ML-243

C4813-5 5 mg
EUR 219.6

ML-097

C4821-5 5 mg
EUR 219.6
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.

ML-336

C3581-5 5 mg
EUR 354
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.

ML-211

C3647-5 5 mg
EUR 208.8
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.

ML-265

C4181-5 5 mg
EUR 216
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.

ML-178

C4287-5 5 mg
EUR 174
Description: ML-178 is a novel and selective S1P4 activator with EC50 value of 46.3 nM [1]. ML-178 is a novel and selective S1P4 activator.

ML 228

A4508-5 5 mg
EUR 236.4
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.

ML 141

B9014-5 5 mg
EUR 195.6

ML-216

B2666-5 5 mg
EUR 199.2

ML-210

B2710-5 5 mg
EUR 199.2

ML-277

B2730-5 5 mg
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B4926-5 5 mg
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calibration solution 50 ml 0.1 m kcl

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B062 ea
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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).