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.

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

TMEXO-1 100 reactions
EUR 583

Exosome precipitation from Plasma and Serum

EPStep-PS 5 ml
EUR 297

ExoQuick Exosome RNA Column Purification kit

EQ808A-1 20 preps
EUR 402

ExoQuick-LP for lipoprotein pre-clear & exosome isolation

EXOLP5A-1 5 reactions
EUR 533

Magnetic Beads (DNA) 5 mL

P920-5 - Ask for price

Proteinase K, Recombinant, 20 mg/ml Solution, Molecular Grade

9211-5
EUR 164

Dispase Solution, 1mg/ml

CA092-010 100ml
EUR 112

Transferrin Solution, 10mg/ml

CA205-005 5x1ml
EUR 115

cleaning solution 500 ml

B530 ea
EUR 30

Kanamycin solution (50mg/ml)

MN800A-1 20 ml
EUR 127

Gentamicin Solution, Gentamicin Solution, 50 mg/mL

CCM1123-010 10 mL
EUR 64.05

ExoQuick PLUS Exosome Purification Kit - for serum and plasma

EQPL10A-1 10 reactions
EUR 579

BSA Blocking Solution, 2 mL

BLK-002 2 mL
EUR 88.51
Description: The BSA solution is blocking gold nanoparticles following conjugation reactions.

RNase A Solution (10mg/ml)

RB0474 1ml
EUR 55.22

BSA Blocking Solution, 10 mL

BLK-010 10 mL
EUR 168.58
Description: The BSA solution is blocking gold nanoparticles following conjugation reactions.

Sodium Selenite Solution,100mg/ml

CA056-010 100ml
EUR 101

DNase I Solution, 1mg/ml

CA095-005 5x1ml
EUR 138

Lentivirus Concentration Solution (100 mL)

P904C - Ask for price

Retrovirus Concentration Solution (100 mL)

P905C - Ask for price

Chloramphenicol Solution (50 mg/ml)

C090-10ML 10 mL
EUR 59

Chloramphenicol Solution (50 mg/ml)

C090-10x10ML 10 x 10 mL
EUR 291

pepsine cleaning solution 250 ml

B231 ea
EUR 45

pepsine cleaning solution 1000 ml

B232 ea
EUR 99

100 ML 45% GLUCOSE SOLUTION

25-037-CI 100 mL/pk
EUR 46
Description: Media Catalog; Cell Culture Reagents

Albumin, Bovine 20mg/ml solution

CH006 1.0 ml
EUR 142

Albumin, Bovine 20mg/ml solution

CH007 5.0 ml
EUR 212

ExoQuick-TC PLUS Exosome Purification Kit - for tissue culture media

EQPL10TC-1 10 reactions
EUR 579

Hematoxylin Solution (5%)

HSV250 250 ml
EUR 133

Hematoxylin Solution (5%)

HSV500 500 ml
EUR 183

Hematoxylin Solution (5%)

HSV999 1000 ml
EUR 277

Sodium chloride 5 M, 1000 ml

12-9191-5 1000 ml
EUR 96

Exosome Component 5 Protein

20-abx260873
  • EUR 3418.00
  • EUR 328.00
  • EUR 230.00
  • 1 mg
  • 25 ug
  • 5 ug

10 ML GENTAMICIN SULFATE, LIQUID 50 MG/ML SOLUTION

30-005-CR 10 mL/pk
EUR 433
Description: Media Catalog; Cell Culture Reagents

50 ML KANAMYCIN SULFATE, LIQUID, 5,000 µG/ML SOLUTION

30-006-CF 50 mL/pk
EUR 98
Description: Media Catalog; Cell Culture Reagents

ExoQuick Exosome Isolation and RNA Purification kit (for Serum and Plasma)

EQ806A-1 20 preps
EUR 554

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

EQ806TC-1 10 preps
EUR 554

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

RA806A-1 20 preps
EUR 554

Linear Poly Acrylamide Solution (5mg/ml)

LK548 1ml
EUR 63.05

Ethidium Bromide, 10mg/ml Stock Solution

E0205-001 10ml
EUR 88

Ethidium Bromide, 10mg/ml Stock Solution

E0205-002 20ml
EUR 128

Fibronectin, Bovine, Stabilized Solution , 1mg/ml

CA082-001 1ml
EUR 313

Fibronectin, Human, Stabilized Solution, 1mg/ml

CA083-001 1ml
EUR 327

Vitronectin , Human , Stabilized Solution, 0.5mg/ml

CA085-050 50ul
EUR 209

Papain Solution In PBS, 1mg/ml

CA093-002 20ml Ask for price

Insulin Solution, 1mg/ml , Animal Free

CA204-005 5x1ml
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7-AAD solution, 1 mg/mL

40084 1mL
EUR 212
Description: Minimum order quantity: 1 unit of 1mL

buffer solution 50 ml 4.00 ph

B004 ea
EUR 21

buffer solution 50 ml 7.00 ph

B007 ea
EUR 21

buffer solution 50 ml 10.00 ph

B010 ea
EUR 21

calibration solution 50 ml 200ms/cm

B066 ea
EUR 24

buffer solution 500 ml 4.00 ph

B504 ea
EUR 30

buffer solution 500 ml 7.00 ph

B507 ea
EUR 30

buffer solution 500 ml 10.01 ph

B510 ea
EUR 30

buffer solution 500 ml 12.00 ph

B512 ea
EUR 30

calibration solution 500 ml 200ms/cm

B566B ea
EUR 45

500 ML L-GLUTAMINE 200MM SOLUTION

25-005-CV 500 mL/pk
EUR 97
Description: Media Catalog; Cell Culture Reagents

100 ML MEM VITAMINS 100X SOLUTION

25-020-CI 100 mL/pk
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Description: Media Catalog; Cell Culture Reagents

100 ML SODIUM BICARBONATE, 7.5% SOLUTION

25-035-CI 100 mL/pk
EUR 58
Description: Media Catalog; Cell Culture Reagents

Sodium hydroxide powder 5 M, 1000 ml

12-9183-5 1000 ml
EUR 122

Exosome isolation Solution - Serum/ Plasma (easy version of P101, 95% pure exosome)

P101EZ - Ask for price

ExoPure? 0.4 micron Immunobeads (Overall Exosome Isolation, cell media, 5 reactions)

M1030-5
EUR 664

ExoPure? 1.0 micron Immunobeads (Overall Exosome Isolation, cell media, 5 reactions)

M1031-5
EUR 664

TumorExoPure? 0.4 micron Immunobeads (Tumor-derived Exosome Isolation, biofluids, 5 reactions)

M1032-5
EUR 664

TumorExoPure? 1.0 micron Immunobeads (Tumor-derived Exosome Isolation, biofluids, 5 reactions)

M1033-5
EUR 664

MouseExoPure? 0.4 micron Immunobeads (Overall Exosome Isolation, mouse biofluids, 5 reactions)

M1034-5
EUR 648

MouseExoPure? 1.0 micron Immunobeads (Overall Exosome Isolation, mouse biofluids, 5 reactions)

M1035-5
EUR 648

Exosome binding buffer for ELISAs (20 ml)

EXOEL-BB-20 20 ml
EUR 219

ECGS/5 mL

C0010-01 RT 500 mL Bottle
EUR 183

Sodium Metabisulfite Solution (5%)

SME125 125 ml
EUR 69

Sodium Metabisulfite Solution (5%)

SME3800 1 Gal.
EUR 266

Sodium Metabisulfite Solution (5%)

SME500 500 ml
EUR 95

Sodium Metabisulfite Solution (5%)

SME999 1000 ml
EUR 122

Silver Nitrate Solution (5%)

SNV125 125 ml
EUR 101

Silver Nitrate Solution (5%)

SNV500 500 ml
EUR 231

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STB125 125 ml
EUR 63

Sodium Thiosulfate Solution (5%)

STB500 500 ml
EUR 96

Sodium Thiosulfate Solution (5%)

STB999 1000 ml
EUR 140

Potassium Ferrocyanide Solution (5%)

PFB3800 1 Gal.
EUR 277

Potassium Ferrocyanide Solution (5%)

PFB500 500 ml
EUR 84

Potassium Ferrocyanide Solution (5%)

PFB999 1000 ml
EUR 111

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PPG030 30 ml
EUR 66

Potassium Permanganate Solution (5%)

PPG125 125 ml
EUR 88

HSA Solution 5% (cGMP)

AK8969-0100 100mL Ask for price

Imidazole (5 M) Solution

B1014-100
EUR 131

Imidazole (5 M) Solution

B1014-500
EUR 305

DNA Precipitation Buffer

D106 55 ml
EUR 175
Description: DNA Precipitation Buffer by Cygnus Technologies is available in Europe via Gentaur.

TMB for Precipitation

42R-TB100x 1000 ml
EUR 538
Description: High sensitivity TMB Substrate for Precipitation assays

Exosome isolation Solution - Cell Media/ Urine (easy version of P100, 95% pure exosome)

P100EZ - Ask for price

Exosome isolation Solution - Serum/ Plasma (easy version of P101, 95% pure exosome) sample

P101EZ--sample - Ask for price

ML-336

C3581-5 5 mg
EUR 295
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 174
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 180
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 145
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-356

C3354-5 5 mg
EUR 395
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
EUR 163
Description: ML264, a potent and selective inhibitor of kruppel-like factor 5 (KLF5), inhibits the growth of colorectal cancer.

ML-354

C3058-5 5 mg
EUR 145
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 240

B4926-5 5 mg
EUR 171

ML 349

B4934-5 5 mg
EUR 206

ML 213

B5628-5 5 mg
EUR 126

ML 204

B5696-5 5 mg
EUR 152

ML 281

B5750-5 5 mg
EUR 206

ML 289

B5783-5 5 mg
EUR 151

ML 228

A4508-5 5 mg
EUR 197
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-291

C5358-5 5 mg
EUR 168
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-031

C5718-5 5 mg
EUR 183
Description: ML-031 is an agonist of S1P2 with EC50 value of 1 ?M [1].

ML 239

A8719-5 5 mg
EUR 139
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 277

B7711-5 5 mg
EUR 183

ML 141

B9014-5 5 mg
EUR 163

ML-193

C4531-5 5 mg
EUR 180
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 183

ML-191

C4786-5 5 mg
EUR 183

ML-233

C4788-5 5 mg
EUR 183

ML-335

C4792-5 5 mg
EUR 158

ML-030

C4805-5 5 mg
EUR 183

ML-098

C4807-5 5 mg
EUR 183

ML-243

C4813-5 5 mg
EUR 183

ML-097

C4821-5 5 mg
EUR 183
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-240

B1142-5
EUR 185

ML-365

B2076-5
EUR 218

ML-35

B2077-5
EUR 218

ML-162

B2304-5
EUR 414

ML-216

B2666-5 5 mg
EUR 166

ML-210

B2710-5 5 mg
EUR 166

ML-277

B2730-5 5 mg
EUR 313

ML-193

B3016-5 5 mg
EUR 176

ML-354

9415-5
EUR 142

ML-323

2658-5
EUR 142

ML-324

2763-5
EUR 359

AXYPREP MAG DYECLEAN KIT- 5 ML - 800 PREPS

MAG-DYECL-5 1/pk
EUR 211
Description: Bioscience Mag Beads; Magnetic Dye

MouseExoPure? 0.4 micron Immunobeads (Overall Exosome Isolation, mouse cell media, 5 reactions)

M1036-5
EUR 648

MouseExoPure? 1.0 micron Immunobeads (Overall Exosome Isolation, mouse cell media, 5 reactions)

M1037-5
EUR 648

ExoPure? 1.0 micron Immunobeads (Overall Exosome Isolation, plasma, urine, serum, 5 reactions)

M1039-5
EUR 664

Exosome Component 5 (EXOSC5) Antibody

20-abx112414
  • EUR 732.00
  • EUR 398.00
  • 150 ul
  • 50 ul

Exosome Component 5 (EXOSC5) Antibody

abx232905-100ug 100 ug
EUR 509

Exosome Component 5 (EXOSC5) Antibody

20-abx302857
  • EUR 411.00
  • EUR 1845.00
  • EUR 599.00
  • EUR 182.00
  • EUR 300.00
  • 100 ug
  • 1 mg
  • 200 ug
  • 20 ug
  • 50 ug

ExoQuick TC 10ml

EXOTC10A-1 10 ml
EUR 399

ExoQuick TC 50ml

EXOTC50A-1 50 ml
EUR 1222

Fetuin Solution, 1mg/ml in DMEM-F12

CA065-005 5x1ml
EUR 97

Puromycin Solution, 10mg/ml , Cell Cultere Tested

CR026-001 10ml
EUR 201

PBS solution pH 7.4, 1000 ml/bottle

10-9402-10 1000 ml/bottle
EUR 161

calibration solution 50 ml 0.01 m kcl

B060 ea
EUR 21

calibration solution 50 ml 0.1 m kcl

B061 ea
EUR 21

calibration solution 50 ml 1 m kcl

B062 ea
EUR 21

redox standard solution 50 ml 124 mv

B071 ea
EUR 22

redox standard solution 50 ml 358 mv

B072 ea
EUR 22

calibration solution 500 ml 0.01 m kcl

B560 ea
EUR 30

calibration solution 500 ml 0.1 m kcl

B561 ea
EUR 30

calibration solution 500 ml 1 m kcl

B562 ea
EUR 30

redox standard solution 500 ml 124 mv

B571 ea
EUR 36

redox standard solution 500 ml 358 mv

B572 ea
EUR 36

X-Gal Solution, 40 mg/ml concentrate

X0321-005 5X1ml
EUR 115

X-Gal Solution, 40 mg/ml concentrate

X0321-020 20ml
EUR 135

CometAssay Lysis Solution (2 x 500 mL)

4250-050-01 1 L
EUR 183

dsGreen Gel Staining Solution, 10000×, 0.5 mL

10010 0.5 mL
EUR 89

dsGreen Gel Staining Solution, 10000×, 1 mL

20010 1 mL
EUR 134

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).