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Integrating automated liquid dealing with within the separation workflow of extracellular vesicles enhances specificity and reproducibility | Journal of Nanobiotechnology

Pattern assortment and crude extract preparation

Blood plasma

Venous blood from breast most cancers sufferers was collected utilizing citrate blood assortment tubes (455,322, Greiner Bio-one). Platelet-depleted plasma was ready by two serial centrifugations at 2500 g for 15 min at room temperature. All blood samples have been first characterised (full blood rely) utilizing the hematology analyzer (XP-300, Sysmex). All blood samples have been processed inside 120 min after blood assortment and platelet-depleted plasma was saved as 1 mL aliquots at −80 °C. Affected person traits are summarized in Extra file 1: Fig. S1A. Crude extracts from whole blood plasma have been ready utilizing SEC columns with Sepharose CL-2B as beforehand described [10] (Fig. 1A). A SEC column was ready by putting a nylon web with 20 μm pore measurement (NY2002500, Merck Millipore) on the underside of a ten mL syringe (3SYR-10ML, Romed), adopted by stacking of 10 mL pre-washed Sepharose CL-2B (17,014,001, GE Healthcare). On prime of 1 SEC column, 2 mL blood plasma was loaded adopted by elution and assortment of 6 sequential 1 mL eluate fractions. Following SEC, eluted fractions 4–5–6 have been pooled and concentrated to 1 mL utilizing a ten kDa centrifugal filter (Amicon Extremely-2 mL, UFC201024, Merck Millipore) (known as the crude extract). The crude extracts have been pooled and aliquots of 1 mL have been saved at −80 °C [20].


Urine from wholesome volunteers was collected and crude extracts have been ready as beforehand described [8]. Urine samples have been centrifuged for 10 min at 1000 g and 4 °C. Cell-free urine supernatants have been collected (leaving roughly 0.5 cm urine above the cell pellet). Cell-free urine samples (50 mL) have been concentrated to 800 µL utilizing a ten kDa centrifugal filter machine (Centricon Plus-70, UFC701008, Merck Millipore) (Fig. 1A) and saved at −80 °C.

All samples have been collected in compliance with the Moral Committee from Ghent College Hospital (approval EC/2014/0655) and related pointers.

EV separation by density gradient centrifugation

Operators and automatic workstation

Guide density gradient preparation and fraction assortment was carried out by operators utilizing a P1000 single channel pipette. The operators have been divided in two teams based mostly on their expertise. Skilled operators have been outlined as having ready greater than 15 density gradients. Inexperienced operators had ready one to 5 gradients most. All inexperienced operators have been educated by the identical teacher within the process by demonstration of the method, obtained pipetting method coaching, and have been guided throughout the preparation and assortment process.

Robotic-assisted density gradient preparation and fraction assortment was carried out utilizing the Biomek 4000 laboratory automation workstation (Beckman Coulter, A99749) with a custom-made script as beforehand described [10] (particulars are supplied within the Extra file 1). The Biomek 4000 automation workstation has 12 deck positions and may pipet 1 µL as much as 1000 µL by liquid-level sensing. To make sure sterility throughout the procedures, the automated liquid handler was outfitted with a positive-pressure HEPA enclosure. The workstation was used for the preparation of density gradients, pattern loading, and assortment of density gradient fractions.

Preparation of top-down and bottom-up OptiPrep density gradients

OptiPrep (60% (w/v) aqueous iodixanol answer, AXS-1,114,542, Axis-Protect) density gradients have been ready as beforehand described [9, 10]. Options of 5, 10, 20, and 40% iodixanol have been made by mixing acceptable volumes of homogenization buffer (0.25 M sucrose (S0389, Sigma-Aldrich), 1 mM EDTA (1,084,180,100, Merck Millipore), 10 mM Tris (103,154 M, VWR) – HCL (44,921.K2, Alfa Aesar) (pH 7.4)) and iodixanol working answer. This working answer was ready by combining a working answer buffer (0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl (pH 7.4)) and a inventory answer of OptiPrep. rEV have been generated by transfection of HEK293T cells (CRL-11,268, ATCC) with gag-EGFP DNA adopted by rEV separation from conditioned medium utilizing density gradient centrifugation as described beforehand [29, 39].

A discontinuous top-down (TD) OptiPrep density gradient was made by layering 4 mL of 40%, 4 mL of 20%, 4 mL of 10% and three.5 mL of 5% iodixanol options on prime of one another in a 16.8 mL open prime polyallomer tube (337,986, Beckman Coulter). 1 mL crude extract from blood plasma or phosphate-buffered saline (PBS, TMS-012-A, Merck Millipore) spiked with 1.85 × 1010 rEV (as quantified by fluorescent nanoparticles monitoring evaluation (fNTA) was overlaid on prime of the gradient (Fig. 1B). (r)EV suspension was made by resuspending 800 µL crude extract from urine or PBS spiked with 1.85 × 1010 rEV (as quantified by fNTA) in 3.2 mL working answer, acquiring a 40% iodixanol suspension. A discontinuous bottom-up (BU) density gradient was ready by overlaying 4 mL (r)EV suspension with 4 mL 20%, 4 mL 10% and three.5 mL 5% iodixanol options, and 1 mL PBS (Fig. 1B).

TD and BU density gradients have been centrifuged for 18 h at 100,000 g and 4 °C (SW 32.1 Ti rotor, Beckman Coulter).

The strategy of handbook making ready density gradients is described in Fig. 1C, Extra file 1: Field S1, and Extra file 2: Video S1.

For the automated preparation of density gradients, the single- and eight-channel P1000 pipetting instruments, tip packing containers (B01122, Beckman Coulter), pre-cooled iodixanol options reservoir, pre-cooled tube rack with the centrifuge tube(s), and pattern rack have been positioned in one of many deck positions of the workstation (Fig. 1D). For density gradient preparation the eight-channel MP1000 device was used (Extra file 3: Video S2), and for pattern loading the single-channel P1000SL device (Extra file 4: Video S3). The custom-made script permits the preparation of as much as eight density gradients inside one run.

Assortment of density gradient fractions

After ultracentrifugation, density gradient fractions of 1 mL have been collected from prime to backside manually or by the liquid handler.

The strategy of handbook fraction assortment is described in Fig. 1C, Extra file 1: Field S1, and Extra file 5: Video S4.

The automated fraction assortment requires the single-channel P1000 pipetting device, tip field(es), pre-cooled tube rack with density gradient(s), and fraction rack(s) (Fig. 1D, Extra file 6: Video S5).

EV restoration by ultracentrifugation or size-exclusion chromatography

To carry out LC-MS/MS, ultracentrifugation was most popular as probably the most sensible methodology to take away OptiPrep from particular person EV-enriched density fractions from blood plasma and urine in a reproducible approach, as beforehand described [10]. Density fractions 9 and 10 have been individually transferred to centrifuge tubes. 14 mL of pre-cooled PBS was added to every pattern and the answer was blended within the tube by pipetting up and down. The tubes have been centrifuged for 3 h at 100,000 g and 4 °C (SW 32.1 Ti rotor, Beckman Coulter). After ultracentrifugation, the supernatant was discarded leaving 50 µL on the backside of the tube. The pellet was diluted to 100 µL with pre-cooled PBS. To stop lack of EV sticking to the underside of the tube, the EV pellet was straight lysed within the tube. Lysates have been ready by mixing samples with SDT-lysis buffer (2% SDS (436143-25G, Sigma-Aldrich), 500 mM Tris (103,154 M, VWR) – HCL (44,921.K2, Alfa Aesar) (pH 7.6), 0.5 M dithiothreitol (39759.02, Serva)) at a 4:1 pattern to buffer ratio. The pellet was pipetted up and down and the underside of the tube was rinsed with SDT-lysis buffer. The lysates have been collected and incubated at 95 °C for five min. Lysates have been saved at − 80 °C till processing for LC-MS/MS.

To carry out transmission electron microscopy (TEM), EV have been separated from pooled density fractions 9–10 obtained from blood plasma and urine samples by together with a second SEC, following beforehand talked about protocol except acknowledged in any other case. From this second SEC, eluted size-based fractions 4-5-6-7 have been pooled and concentrated to 100 µL utilizing a ten kDa centrifugal filter (Amicon Extremely-2 mL, UFC201024, Merck Millipore) and saved at −80 °C.

Density measurement

The density of the density-gradient fractions was calculated utilizing a regular curve of the absorbance values at 340 nm (SpectraMax Paradigm, Molecular Units) of 1:1 aqueous dilution of 5, 10, 20 and 40% iodixanol options. This customary curve was used to find out the density of fractions collected from a management gradient overlaid with 1 mL of PBS.

Interface mixing

To find out interface mixing, the picture of coloured check density gradients ready by an inexperienced, skilled, and automatic operator was analyzed utilizing ImageJ software program model 1.53. Every 10% iodixanol layer was individually circumscribed with the oblong area of curiosity (ROI) choice. Colours have been transformed to binary. Profile plots of the ROIs have been generated. Space measurement of the peaks (corresponding with the spilling of the 5% and 20% iodixanol answer within the 10% layer) was carried out with the wand device.

Fluorescent nanoparticle monitoring evaluation

Fractions of rEV spiked density gradients have been analyzed by fluorescent nanoparticle monitoring evaluation (fNTA) utilizing a NanoSight LM10-HS microscope (Malvern Devices Ltd) outfitted with a 488 nm laser, a further 500 nm longpass filter and an automated syringe pump system (infusion pace: 20) (Fig. 1E). For every evaluation, three movies of 60 s have been recorded and analyzed with digital camera stage 16 and detection threshold 3. Temperature was monitored throughout recording. Recorded movies have been analyzed with the NTA Software program model 3.3. For optimum measurements, samples have been diluted with PBS till particle focus was inside the focus vary for the NTA Software program (3 × 108-109 particles/mL). For restoration calculations the variety of fluorescent particles was measured earlier than spiking.

Anti-p24 ELISA

Gag-EGFP protein concentrations in fractions of rEV spiked density gradients have been decided with the anti-p24 ELISA equipment Innotest HIV antigen mAb (80,563, Fujirebio) (Fig. 1E) in line with the producer’s directions. For restoration calculations a rEV customary curve, from the identical batch as used for spiking, was included starting from 1 × 106-107 fluorescent particles as beforehand measured with fNTA.

Western blot

Protein concentrations of rEV have been measured, after lysis with 0.2% SDS (436143-25G, Sigma-Aldrich), with the Qubit Protein Assay (ThermoFisher) and Qubit fluorometer 3.0 following producer’s directions. For protein evaluation, samples have been dissolved in lowering pattern buffer (0.5 M Tris-HCl [pH 6.8], 40% glycerol, 9.2% SDS, 3% 2-mercaptoethanol, 0.005% bromophenol blue) and boiled at 95 °C for five min. Proteins have been separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes have been blocked for 30 min in blocking buffer (5% non-fat milk in PBS with 0.5% Tween-20) and incubated in a single day at 4 °C with main antibodies (mouse monoclonal anti-ALIX (1:1000, #2171); rabbit monoclonal anti-CD9 clone D3H4P (1:1000, #13403S) (Cell Signaling Expertise); and mouse monoclonal anti-flotillin-1 (1:1000, #610,820) (BD Biosciences)). Secondary antibodies (sheep anti-mouse horseradish peroxidase-linked antibody (1:3000, #NA931V) and donkey anti-rabbit horseradish peroxidase-linked antibody (1:4000, #NA934V) (GE Healthcare Life Sciences)) have been added for 60 min at room temperature after intensive washing with blocking buffer. After closing washing, chemiluminescence substrate (WesternBright Sirius, Advansta) was added and imaging was carried out utilizing Proxima 2850 Imager (IsoGen Life Sciences).

Protein measurements

Protein concentrations of the lysed EV preparations obtained from blood plasma and urine samples have been measured utilizing the fluorometric Qubit Protein Assay (ThermoFisher) and the Qubit Fluorometer 3.0 (ThermoFisher) in line with the producer’s directions.


EV preparations obtained from blood plasma and urine samples have been processed for LC-MS/MS by filter-aided pattern preparation (FASP) [40] (Fig. 1E). After thawing and clarification by centrifugation (16,000 g for five min), lysates have been blended with 300 µL UA (8 M urea (U5128, Sigma-Aldrich), 0.1 M Tris-HCl (pH 8.5)) in a Microcon-10 kDa centrifugal filter machine (MRCPRT010, Merck Millipore). Filters have been centrifuged twice (14,000 g for 40 min at 20 °C) with the addition of 200 µL UA in between. Proteins have been alkylated by addition of 100 µL IAA answer (0.05 M iodoacetamide (I1149, Sigma-Aldrich) in UA buffer) and incubated for 30 min at room temperature, adopted by centrifugation. Samples have been handled twice by addition of 100 µL UA and centrifugation. Subsequently, samples have been twice handled by addition of 100 µL DB buffer (1 M urea, 0.1 M Tris-HCl (pH 8.5) and centrifugation. Filter items have been transferred to new assortment tubes and proteins have been resuspended in 40 µL DB with Trypsin/Lys-C combine (V5073, Promega) for in a single day proteolytic digestion at 37 °C. Digests have been collected by addition of 100 µL DB and centrifugation for 15 min at 14,000 g. This step was repeated as soon as. Collected peptides have been acidified with 1% trifluoroacetic acid to a pH of two–3, adopted by desalting with Peptide Cleanup C18 Spin Tubes (5188 − 2750, Aligent). Desalted peptides have been vacuum dried, dissolved in 0.1% formic acid and analyzed by LC-MS/MS. Equal quantities of peptides of every pattern have been loaded on a nanoflow HPLC system (Simple- nLC1000, Thermo Fisher Scientific) coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) outfitted with a nano-electrospray ionization supply. The cell section consisted of 0.1% formic acid (solvent A) and acetonitrile/water (95:5 (v/v)) with 0.1% formic acid (solvent B). The peptides have been separated with a 40 min gradient from 8 to 35% of solvent B. Earlier than the tip of the run, the proportion of solvent B was raised to 100% in 2 min and stored there for 8 min. Full MS scan over the mass-to-charge (m/z) vary of 300–1750 with a decision of 120,000, adopted by information dependent acquisition with an isolation window of two.0 m/z and a dynamic exclusion time of 30 s was carried out. The highest 12 ions have been fragmented by increased power collisional dissociation (HCD) with a normalized collision power of 27% and scanned over the m/z vary of 200–2000 with a decision of 15,000. After the MS2 scan for every of the highest 12 ions had been obtained, a brand new full mass spectrum scan was acquired, and the method repeated till the tip of the 50 min run. Three repeated runs per pattern have been carried out. Tandem mass spectra have been searched utilizing the MaxQuant software program (model in opposition to a database containing reviewed sequences of homo sapiens of UniProtKB launch 2019_11. Peptide-spectrum-match- and protein-level false discovery charges have been set at 0.01. Carbamidomethyl (C), as a set modification, and oxidation (M) and acetylation of the protein N-terminus as dynamic modifications have been included. A most of two missed cleavages was allowed. The LC-MS profiles have been aligned, and the identifications have been transferred to non-sequenced or non-identified MS options in different LC-MS runs (matching between runs). The protein was decided as detected within the pattern if its identification had been derived from at the least two distinctive peptide identifications. Filtering for contaminating proteins, reverse identification and identification by web site was used. Label-free quantification (LFQ) was carried out utilizing the MaxLFQ algorithm built-in within the MaxQuant software program.

Proteomic information evaluation

Recognized proteins have been analyzed and visualized utilizing Perseus software program model [41]. Proteins exhibiting legitimate values in at the least 70% of at the least one group have been chosen. Reverse database hits and potential contaminant proteins have been eliminated. Lacking values have been imputed from the noticed regular distribution of intensities. LFQ intensities have been normalized utilizing the Width adjustment methodology in Perseus. For chosen analyses, the normalized LFQ intensities have been log2 reworked. Coefficient of variation (CV) evaluation was based mostly on the 100 highest quantified proteins inside every pattern sort. Principal part evaluation (PCA) was carried out utilizing the Perseus software program. Unsupervised hierarchical clustering warmth maps, utilizing 1-Pearson correlation, have been generated utilizing the Morpheus device. Evaluation of similarities (anosim) was carried out utilizing Past3 software program [42]. The Vesiclepedia database was explored to determine the 100 most typical EV-associated proteins [43]. Quantitative expression profile based mostly useful enrichment evaluation was carried out FunRich software program model 3.1.3 [44].

Transmission electron microscopy

EV preparations obtained from blood plasma and urine have been qualitatively analyzed with transmission electron microscopy (TEM) (Fig. 1E). Samples have been deposited on a formvar coated grids stabilized with evaporated carbon movie and glow discharged earlier than pattern software (AGS162-3 H, Agar Scientific). Impartial uranyl acetate (2% in AD) (21447-25, Polysciences) was used for staining after which grids have been coated with 2% methyl cellulose (M7027, Sigma-Aldrich) / uranyl acetate (0,4%) answer. These grids have been examined utilizing a Tecnai G2 Spirit transmission electron microscope (Thermo Fisher Scientific FEI) operated at 100 kV and pictures have been captured with a Quemesa charge-coupled machine digital camera (Olympus Gentle Imaging Options).

Info on density measurement, interface mixing, and characterization strategies (Fig. 1E) of rEV (fNTA and anti-p24 ELISA) and EV (mass-spectrometry based mostly proteomics and TEM) is supplied within the Extra file 1.

Statistical evaluation

Information evaluation and visualization was carried out utilizing GraphPad Prism model 8 (GraphPad Software program). Information are expressed as median with interquartile vary (IQR). Correlations have been calculated utilizing the Pearson product-moment (r). Variations of imply ranks have been evaluated by Mann Whitney U check and variations of variance by F-test of equality of variances. P-values smaller than 0.05 have been thought-about statistically vital.



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