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.

PEG-it Virus Precipitation Solution (250 mL aliquot)
LV825A-1 250 ml
EUR 777
  • Category: Lentiviral Technology
PEG-it Virus Precipitation Solution (100 mL aliquot)
LV810A-1 100 ml
EUR 382
  • Category: Lentiviral Technology
ExoQuick-CG cGMP-grade Exosome Isolation reagent (50 ml)
EXOCG50A-1 50 ml
EUR 1181
  • Category: Exosomes
Exosome precipitation from Plasma + Thrombin
EPStep-T 5 ml
EUR 336
Thrombin Plasma prep for Exosome precipitation (500 ul at 500U/mL)
TMEXO-1 100 reactions
EUR 583
  • Category: Exosomes
ExoQuick Exosome RNA Column Purification kit
EQ808A-1 20 preps
EUR 402
Exosome precipitation from Plasma and Serum
EPStep-PS 5 ml
EUR 297
ExoQuick-LP for lipoprotein pre-clear & exosome isolation
EXOLP5A-1 5 reactions
EUR 533
  • Category: Exosomes
ExoQuick PLUS Exosome Purification Kit - for serum and plasma
EQPL10A-1 10 reactions
EUR 579
  • Category: Exosomes
Magnetic Beads (DNA) 5 mL
P920-5 NULL
EUR 0
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
  • Category: Minicircle Technology
Proteinase K, Recombinant, 20 mg/ml Solution, Molecular Grade
9211-5
EUR 164
Gentamicin Solution, Gentamicin Solution, 50 mg/mL
CCM1123-010 10 mL
EUR 64.05
  • Product category: Culture Media
ExoQuick-TC PLUS Exosome Purification Kit - for tissue culture media
EQPL10TC-1 10 reactions
EUR 579
  • Category: Exosomes
Sodium Selenite Solution,100mg/ml
CA056-010 100ml
EUR 101
DNase I Solution, 1mg/ml
CA095-005 5x1ml
EUR 138
pepsine cleaning solution 250 ml
B231 ea
EUR 45
pepsine cleaning solution 1000 ml
B232 ea
EUR 99
BSA Blocking Solution, 2 mL
BLK-002 2 mL
EUR 88.51
  • Product Category: Gold Ribbon, Gold Conjugation
Description: The BSA solution is blocking gold nanoparticles following conjugation reactions.
BSA Blocking Solution, 10 mL
BLK-010 10 mL
EUR 168.58
  • Product Category: Gold Ribbon, Gold Conjugation
Description: The BSA solution is blocking gold nanoparticles following conjugation reactions.
Chloramphenicol Solution (50 mg/ml)
C090-10ML 10 mL
EUR 59
Chloramphenicol Solution (50 mg/ml)
C090-10x10ML 10 x 10 mL
EUR 291
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
Lentivirus Concentration Solution (100 mL)
P904C NULL
EUR 0
Retrovirus Concentration Solution (100 mL)
P905C NULL
EUR 0
RNase A Solution (10mg/ml)
RB0474 1ml
EUR 55.22
  • Product category: Proteins/Enzymes/Endonucleases
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
  • Category: MicroRNA Tools
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
  • Shipped within 5-10 working days.
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
Exosome isolation Solution - Serum/ Plasma (easy version of P101, 95% pure exosome)
P101EZ NULL
EUR 0
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
EUR 115
calibration solution 500 ml 200ms/cm
B566B ea
EUR 45
buffer solution 500 ml 2.00 ph
B502 ea
EUR 30
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
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
Ethidium Bromide, 10mg/ml Stock Solution
E0205-001 10ml
EUR 88
Ethidium Bromide, 10mg/ml Stock Solution
E0205-002 20ml
EUR 128
7-AAD solution, 1 mg/mL
40084 1mL
EUR 212
Description: Minimum order quantity: 1 unit of 1mL
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
EUR 67
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
Linear Poly Acrylamide Solution (5mg/ml)
LK548 1ml
EUR 63.05
  • Product category: PCR Related/Nucleic Acid Co-precipitants
ExoQuick TC 10ml
EXOTC10A-1 10 ml
EUR 399
  • Category: Exosomes
ExoQuick TC 50ml
EXOTC50A-1 50 ml
EUR 1222
  • Category: Exosomes
Sodium hydroxide powder 5 M, 1000 ml
12-9183-5 1000 ml
EUR 122
Exosome binding buffer for ELISAs (20 ml)
EXOEL-BB-20 20 ml
EUR 219
  • Category: Exosomes
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
TMB for Precipitation
42R-TB100x 1000 ml
EUR 538
Description: High sensitivity TMB Substrate for Precipitation assays
DNA Precipitation Buffer
D106 55 ml
EUR 175
  • Product category: DNA and RNA extraction and purification
Description: DNA Precipitation Buffer by Cygnus Technologies is available in Europe via Gentaur.
ECGS/5 mL
C0010-01 RT 500 mL Bottle
EUR 183
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
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
Potassium Permanganate Solution (5%)
PPG030 30 ml
EUR 66
Potassium Permanganate Solution (5%)
PPG125 125 ml
EUR 88
Sodium Thiosulfate Solution (5%)
STB125 125 ml
EUR 63
Sodium Thiosulfate Solution (5%)
STB500 500 ml
EUR 96
Sodium Thiosulfate Solution (5%)
STB999 1000 ml
EUR 140
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
Exosome isolation Solution - Cell Media/ Urine (easy version of P100, 95% pure exosome)
P100EZ NULL
EUR 0
Exosome isolation Solution - Serum/ Plasma (easy version of P101, 95% pure exosome) sample
P101EZ--sample NULL
EUR 0
Exosome Component 5 (EXOSC5) Antibody
20-abx112414
  • EUR 732.00
  • EUR 398.00
  • 150 ul
  • 50 ul
  • Shipped within 5-10 working days.
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
  • Shipped within 5-10 working days.
Exosome Component 5 (EXOSC5) Antibody
abx232905-100ug 100 ug
EUR 509
  • Shipped within 5-12 working days.
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
AXYPREP MAG DYECLEAN KIT- 5 ML - 800 PREPS
MAG-DYECL-5 1/pk
EUR 211
Description: Bioscience Mag Beads; Magnetic Dye
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-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-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-365
B2076-5
EUR 218
ML-35
B2077-5
EUR 218
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-240
B1142-5
EUR 185
ML-323
B1317-5 5 mg
EUR 125
Description: IC50: ML-323 is a highly potent and selective inhibitor of USP1-UAF1 with IC50 value of 76 nM.ML-323 is an inhibitor of USP1-UAF1 with remarkable selectivity and relatively low cytotoxicity to the human cells.
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 277
B7711-5 5 mg
EUR 183
ML 141
B9014-5 5 mg
EUR 163
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-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-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-354
9415-5
EUR 142
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 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-323
2658-5
EUR 142
ML-324
2763-5
EUR 359
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
Fetuin Solution, 1mg/ml in DMEM-F12
CA065-005 5x1ml
EUR 97
redox standard solution 500 ml 124 mv
B571 ea
EUR 36
redox standard solution 500 ml 358 mv
B572 ea
EUR 36
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
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
CometAssay Lysis Solution (2 x 500 mL)
4250-050-01 1 L
EUR 183
PBS solution pH 7.4, 1000 ml/bottle
10-9402-10 1000 ml/bottle
EUR 161
dsGreen Gel Staining Solution, 10000×, 0.5 mL
10010 0.5 mL
EUR 89

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).