The present invention relates to the field of extracellular vesicles isolation from cell culture media or biological fluids.
Extracellular vesicles (EVs) are membranous particles that include exosomes (80-200 nm), microvesicles (100-600 μm) and apoptotic bodies (800-5000 nm). This classification is mainly based on vesicle size, although different mechanisms have been proposed for their biogenesis. In oncology, EVs hold potential to study the modulation of tumor microenvironment and immune surveillance, to capture information and/or biomarkers released from the tumor, or to be exploited as carriers of therapeutics.
Biological and biomedical research is increasingly focused on the role of EVs in different physiological and pathological processes. Therefore, many techniques for EV isolation from biological material have been proposed to date, although many of them are not very efficient or standardizable (Thery C, et al., Curr Protoc Cell Biol, 2006; Gardiner C, et al. Extracell Vesicles 2016).
The techniques so far reported for EV isolation largely relateto the exosome purification, i.e. smaller EVs with a size between 50 and 200 nm. The techniques widely cited are reported as follows:
Ultracentrifugation is now considered the most effective and widely applied (gold-standard) procedure as a primary isolation method (Gardiner C et al, J Extracell Vesicles, 2016; Al-Nedawi K and Read J Methods Mol Biol. 2016), due to the fact that alternative density gradient centrifugations and precipitation techniques use chemical agents interfering with EV yield, composition and integrity; immunoaffinity capture leads to differential isolation of EV subpopulations (hence low heterogeneity) and it is expensive because it involves antibodies; exclusion chromatography requires significant volumes of biological sample giving a very low yield; column- and centrifuge-based systems strongly damage EV integrity.
However, there are critical issues even in ultracentrifugation: they concern laboriousness, important sample volume to be processed and timing (6-12 hours), purity of the resulting obtained sample, instrument used, operator experience, high level of contaminants (protein aggregates) co-sedimenting with EVs, degradation of biomolecules due to the processing time (Lobb et al, J Extracell Vesicles 2015, Gardiner C et al, J Extracell Vesicles, 2016).
At the state of the art, Ni2+-functionalized stationary phases are known (such as Immobilized Metal Ion Affinity Chromatography (IMAC), nickel chelate acceptor beads, Dynabeads) designed for the purification or recognition of recombinant proteins with histidine tag. In these functionalized stationary phases, the positive net charge deriving from functionalization is hardly ever described by manufacturer, reporting instead the stability index at different pH (very variable), which influences binding efficiency in solution to proteins with a broad range of size (from a few to hundreds kDa).
The purpose of the present invention is to provide a suitable functionalized stationary phase, and a related method of functionalizing and exploiting it, as a new instrument for isolating extracellular vesicles (EVs); said method must be faster, less laborious and more efficient than the ultracentrifuge, and presents further advantages compared to current methods.
The present invention provides a stationary phase which can be consisting of magnetic or non-magnetic particles, of micrometric or nanometric size, functionalized with Ni2+ or Al3+ cations and characterized in that it has a positive net charge between 30 and 80 mV, associated with efficiency on heterogeneous EV isolation as shown below.
The stationary phase (e.g. agarose beads) according to the invention allows a rapid and efficient isolation of whole EVs characterized by a wide range of dispersity with a size range between 50 and 2000 nm that therefore allows to capture both exosomes and microvesicles in the biofluids.
In an aspect, the present invention relates to a method for preparing the stationary phase as described above, said method comprising suspending a non-functionalized stationary phase in a saline solution buffered at a physiological pH containing from a minimum of 15 mM to a maximum of 100 mM (based on the stationary phase capacity and in order to obtain the efficiency shown below on the isolation of heterogeneous EVs) of a Ni2+ or Al3+ salt, and in any case according to the capacity of the stationary phase employed and the net positive charge obtained. In an aspect, the present invention relates to a method for isolating EVs secreted by eukaryotic or prokaryotic cells (bacteria) in culture media or biological fluids, said method comprising the use of the stationary phase functionalized with nickel ions as described above. The method of the invention is hereinafter referred to as NBI (nickel-based isolation).
Extracellular vesicles (EVs) present physicochemical properties, such as structure, size, buoyant density, optical properties and electrokinetic potential (zeta potential) depending on their lipidic double layer structure and lipid and protein composition (Yáñez-Mó M et al, J Extracell Vesicles 2015).
The principle of the method of the invention presented here is based on the exploitation of EV electrokinetic potential (hereinafter referred to as ZP) in combination with a stationary phase (agarose, silicon, magnetic beads, etc.) functionalization with nickel ions for their isolation and purification.
Several reports recently published show that in physiological solution of PBS the ZP of extracellular vesicles is between −17 and −35 mV (Rupert D L et al, Biochim Biophys Acta 2017), an index of moderate-good stability and good dispersity (Correia et al., Langmuir, 2004).
The advantages of the NBI method, according to the invention, with respect to the gold standard technique (UC) used for EV purification are:
In an aspect, the present invention also relates to a kit comprising: a container containing a stationary phase as described above.
The stationary phase according to the invention is preferably functionalized with nickel.
The stationary phase is preferably selected from the group consisting of agarose or silicon beads, whether magnetic or non-magnetic, alginate salt matrices, polymers for Immobilized Metal ion Affinity Chromatography (IMAC), nickel chelate acceptor beads, anionic (as styrene divinylbenzene) or carbon (as graphene) styrene polymers. Agarose beads are particularly preferred.
By micrometric size we mean from 0.5 to 1000 μm; by nanometric size we mean from 1 to 500 nm.
The method of the present invention is hereinafter described on the basis of an embodiment with agarose beads of known nominal size (preferably 25-40 μm) functionalized with nickel ions, i.e. positively charged, therefore allowing the binding in a physiological solution to negatively charged nanoparticles and microparticles. The quantity of nickel ions exposed to the concentration of beads used confer to them electrochemical properties resulting in a positive net charge between 30 and 80 mV, stable for at least six months if the beads are stored at 4° C., in physiological phosphate buffer saline (PBS) solution. Any preservatives such as sodium azide or 20% ethanol can be added to the storage solution, after extensive washing in PBS prior to use the stationary phase.
The saline solution at physiological pH is preferably PBS, but may be substituted with any other buffered solution (Trizma Base, HEPES, etc.) not interfering with divalent cations, e.g. nickel, (in transition or stabilized in detectable forms) and buffered at physiological pH 7.4. The buffered saline solution containing Ni2+ ions is preferably further sterilized with 0.2 μm filters.
The agarose beads according to the invention are preferably prepared from non-functionalized beads by incubation in a 15-30 mM solution of a nickel salt (preferably nickel sulphate, or nickel chloride, nickel oxide, or, in the case of functionalization with aluminium, aluminium sulphate, aluminium chloride, aluminium oxides and/or aluminium silicates) in PBS at pH 7.4, sterilized with 0.2 μm filters.
Preferably the mixture of beads in the nickel salt solution is incubated at room temperature (20-25° C.) and in gentle orbital rotation for a minimum of 1-3 minutes.
After incubation the beads are separated from the solution by centrifugation.
The supernatant is removed and the beads washed with a buffered saline solution, preferably sterilized. Preferably, washing may be repeated 2-3 times to remove nickel ion traces and residual counter-ions by suspension, centrifugation and supernatant removal.
After washing the beads are preferably suspended in a volume, preferably equal to that of the beads, of saline solution buffered at physiological pH, preferably sterilized with 0.2 μm filters, and they (hereinafter referred to as CBeads) can be stored at 4° C.
If the stationary phase is (as commercially available) functionalized with Ni2+ or Al3+ cations, before functionalization as described above, it is necessary to subject it to stripping by one or more washing with an aqueous solution supplemented with 200-300 mM NaCl or KCl, 100-300 mM EDTA or EGTA, 300-500 mM Imidazole, or a solution containing cationic chelating agents with a wide pH range (generally between 5 and 8), and one or more washing with bi-distilled water (18.2 MΩ cm−1). The method according to the present invention involves the use of CBeads which can be added dropwise to the surface of a biological liquid preferably clarified by cellular debris by centrifugation at 2800 rcf, collected in tubes of any size and incubated at room temperature for a minimum 30 minutes. CBeads are added in a volumetric ratio of 10-30 μl for each ml of sample, and an excess thereof is empirically established on the basis of the number of particles found in the biological fluid.
The biological liquid can be:
After incubation with the biological sample CBeads+EVs are separated by decantation. A weak centrifugation at a maximum speed of 300 rcf is allowed to speed up the step and, at the same time, to preserve EVs and stationary phase integrity during the NBI procedure.
The detachment of EVs from the CBeads is carried out by adding a solution (from now on defined Elution), freshly prepared in PBS at pH 7.4 before use, obtained by mixing two solutions A and B containing at least 2 different chelating agents.
Said solution A preferably contains 3-6 mM EDTA at pH 8.0; more preferably solution A is PBS supplemented with 3-6 mM EDTA at pH 8.0.
Said solution B preferably contains 30-300 μM sodium citrate; more preferably the solution B is PBS supplemented with 30-300 μM sodium citrate and 50-100 mM NaCl. For both solution A and B, other chelating agents that can be used are Imidazole, DTPA, NTA, amidoxime, molecules designed for the purpose or peptides which can establish covalent and/or competitive binding with Ni2+ and, therefore, promoting a step of EV release from the stationary phase more or less efficient.
Once the solutions A and B have been mixed, a suitable amount of KH2PO4 is added (8 μl of KH2PO4 are added when EDTA is 3.2 mM, NaCl is 60 mM and sodium citrate is 45 μM) or of any other saline solution buffering to the physiological pH 7.4 and at the same time, not interfering with nickel ions and/or with the morphological-biochemical properties of the EVs.
The CBeads+EVs are preferably incubated with at least the same volume of Elution solution, said incubation preferably in orbital rotation for a minimum of 10-20 minutes at 20-37° C., preferably 28° C.
To separate the CBeads from the Elution+EV solution it is preferable to centrifuge in a tilting rotor at a minimum of 300 rcf for about 1 minute. The supernatant containing whole and polydispersed EVs (generally distributed between 50 and 2000 nm), is transferred into preferably low-binding sample tubes.
The above description with reference to agarose beads is valid, with obvious adjustments, for any other stationary phase according to the present invention.
The aforesaid procedure, described in all its steps, may be supported by further modifications or instrumental couplings suitable to contain the stationary phase, of whatever nature according to the biochemical conditions specified above, in conditions of stability or mobility (columns, matrices, filters, etc.) and which allows for a simplification or procedural optimization.
The method described above is compatible with downstream applications such as RNA extraction from EVs, EV precipitation and/or sorting based on antibodies, amplification of nucleic acids contained or adsorbed in EV by PCR (polymerase chain reaction) and technical variants thereof, transfection of EV in eukaryotic cells, EV engineering with nucleic acids, EV exploitation as nucleic acid, peptide or pharmacological agent carriers.
In an aspect, the present invention relates to a kit comprising:
Preferably the kit further comprises:
Preferably, the kit further comprises:
Preferably, the kit can further comprise:
The kit described above wherein preferably:
The present invention will be better understood in light of the following embodiments.
The procedure for beads functionalization is carried out as follows:
The quantity of nickel ions exposed to the beads gives them electrochemical properties resulting in a positive net charge between 40 and 60 mV, stable for at least six months at room temperature, in phosphate buffer saline (PBS) physiological solution.
CBeads can be added dropwise to the surface of a biological liquid (clarified by cellular debris by 2800 rcf centrifugation) collected in tubes of any size and incubated at room temperature for 30 minutes in a volumetric ratio of 20 μl/ml.
Biological fluid may be cell culture medium mostly containing 1.5% fetal bovine serum (FBS)-PBS at pH 7.4 dilution is allowed if FBS percentage is higher; liquid biopsy sample (whole blood or serum or plasma, urine, cerebrospinal fluid, milk, saliva).
EV isolation from the biological sample is carried out as follows:
CBeads are incubated with the biological sample with gentle orbital rotation (300-600 rpm) for 30 minutes at room temperature, at the end of which the tube is stabilized in a vertical position to allow gravity settling or weak centrifugation (100-400 rcf) of the CBeads (7-15 minutes) at the bottom of the tube.
EV purification, i.e. their removal from the beads, is promoted by a solution (defined from now on Elution) prepared a few minutes before use in PBS at pH 7.4, given by mixture of two solutions A and B containing chelating agents.
EV-Elution A: PBS supplement with final of 3.2 mM EDTA pH 8.0.
EV-Elution B: complete PBS with 60 mM NaCl, 45 μM sodium citrate.
Once the EV-Elution A and B buffers are mixed (Elution 1× solution is obtained), 8 μl/ml KH2PO4 are added to the Elution solution just before the EV elution.
Elution solution allows an ion exchange among the elements in solution and promotes a rapid EV separation from the agarose beads, while preserving EV integrity, size and morphology.
EV purification is carried out as follows:
The supernatant, containing whole and polydispersed EVs (generally distributed between 50 and 800 nm), is transferred to low-binding tubes.
NBI was applied to isolate vesicles released from U87 gliomas cells and the particle number with ≥0.5 μm in diameter (as estimated by flow cytometry) is comparable to that obtained using differential UC, unlike few events captured by non-functionalized beads (
In order to analyse particle populations and refine the elution step that allowed EV enrichment in solution, the tunable resistive pulse sensing (TRPS) was systematically used with the qNANO instrument.
In order to evaluate selectivity of elution, competitive tests were performed in a protein enriched system, including recombinant proteins with 6× histidine, a tag known to confer the strongest interaction with Ni2+. The serum-free medium of U87 cells, complemented with raw extracts of DH5a E. coli cells (500 μg/ml) and with different purified proteins (50 μg/ml each, T7p07, 105 kDa, HuR, 36 kDa; YTH, 23 kDa,
Transmission electron microscopy (TEM) (20500× and 87000× magnifications,
To analyse the impact of mechanical forces and salts equilibrium during the NBI procedure, liposomes similar to exosomes were produced at four different mean sizes (149, 177, 196, 202 nm) and a mixture thereof was added in 10 ml of DMEM medium before processing with NBI (
Since biological materials are subjected to different storage conditions, we analysed the turnover of microvesicles stored at 4° C. after purification by NBI or UC (
To evaluate the robustness of NBI in cellular systems, EVs were purified independently from U87 cell mediums seeded at different densities, in triplicate on 6-well plates (
Isolated EVs were then compared with NBI method from MCF-7, PC3, MDA-MB-231 and SH-SY5Y tumor cell lines. In all cases, an equivalent distribution of vesicles of corresponding size was observed, except for SH-SY5Y cells that produced a weaker release of both vesicle populations (
Since EVs are released from many types of blood cells and could be studied as biomarkers, the performance of NBI on liquid biopsies from healthy donors with known counts of corpuscular elements was evaluated (
Immunodeplection of purified vesicles using erythrocyte CD235a marker or platelet CD41a marker reduced the presence of exosomes or microvesicles in solution (
Finally, RNA extracted from 47 donor EVs and converted into cDNA was used to calculate the absolute number of GAPDH mRNA/˜3*109 EV for Droplet digital PCR assay (
The NBI method presented here is also compatible with other technologies used for the ultrasensitive detection of antigens (proteins) that may be present on EV surface. According to the principle of interactions between positive (metals such as nickel or aluminium) and negative (such as EV) net charges, NBI can be coupled to AlphaScreen (Perkin Elmer) technology, using nickel-chelated Acceptor beads and biotinylated antibodies recognized by Donor beads (
The NBI method allows further verification of EV-associated protein presence by western blotting technique, of which experimental result is shown in
In conclusion, NBI is the next-generation instrument for extracellular vesicle isolation.
Materials and Methods
Cell Cultures
U87-MG human glioma cells (ATCC® HTB-14™), SH-SY5Y dineuroblastoma cell lines (ATCC® CRL-2266™) and PC-3 prostatic adenocarcinoma (ATCC® CRL-1435™) were obtained from ATCC bank (American Type Culture Collection). The cell lines of mammary adenocarcinoma MCF7 (ICLC; HTL95021) and MDA-MB-231 (ICLC; HTL99004) were instead provided by the biological bank of the IRCCS Azienda Ospedaliera Universitaria San Martino—IST Istituto Nazionale per la Ricerca sul Cancro. These cells grow adherent, and except for PC-3 cells, which were kept in culture in RPMI 1640 medium, all the other lines were grown in DMEM medium, both added with 10% FBS (v/v), 100 U/ml penicillin+100 ug/ml of streptomycin, 2 mM L-glutamine (Life Technologies, Carlsbad, Calif., USA), and incubated at 37° C., with 5% CO2. To obtain extracellular vesicle containing medium, the cells were initially cultured in whole medium until reaching 75% confluence (usually in 48 hours); subsequently, after having been gently washed twice with PBS, cells were incubated in a FBS-free medium for 24 hours. Cells were plated in different plate and flask formats according to the experiments to be carried out, but the density was kept constant at 3.2±0.2*104/cm2, unless otherwise described in the figure legend.
Before starting the NBI procedure, the collected culture medium was centrifuged at 2800 rcf for 10 minutes and gently transferred into new tubes. For the experiments described in
For cell density experiments in
EV Isolation by Differential Ultracentrifugation
The EVs produced by U87-MG cells cultured in T150 flasks (CLS430823-50EA) were isolated by differential ultracentrifugation in accordance with the protocol described in Di (Vizio et al., Am J Pathol, 2012; 15: 1573-84) with minor modifications. Briefly, after 24 hours incubation in a FBS-free medium, the supernatants were collected in falcon tubes and centrifuged at 2,800 rcf at 4° C. to remove cellular debris. The supernatants were then transferred into ultracentrifuge tubes (Polyallomer Quick-Seal centrifuge tubes 25×89 mm, Beckman Coulter) and centrifuged for 30 minutes at 4° C. at 10000 rcf in an Optima XE-90 (Beckman Coulter) instrument with SW 32 Ti rotor. This step allowed preferential precipitation of microvesicles, which were gently re-suspended in filtered PBS. Then, according to the protocol described in Thèry C. et al. (Curr Protoc Cell Biol. 2006; Chapter 3: Unit 3.22), the collected supernatants were filtered through a 0.22 μm disposable filter (Sarstedt, Numbrecht, Germany) to remove microvesicle contaminants or aggregates, and centrifuged at 100,000 rcf for 70 min at 4° C. to preferentially pellet exosomes. The pellets were re-suspended in filtered PBS. EVs obtained from differential ultracentrifugation were combined and stored at −80° C. or kept at 4° C. before being analysed by TRPS.
NBI reagents (preferably used):
PBS (ThermoFisher, 10010023) filtered with a 0.2 μm disposable filter membrane (used throughout the whole NBI protocol).
NiSO4 [0.1 M] (Sigma, 656895)
NaCl [5 M] (Sigma, 450006)
Sodium citrate [0.2 M] (Sigma, C8532)
EDTA [0.5] M (ThermoFisher, UltraPure pH 8.0, 15575020)
KH2PO4 [1M] (Sigma, P9791)
Stripping buffer: PBS+0.5 M NaCl, 50 mM EDTA pH 8.0
EV-Elution A to volume with PBS with a final concentration of 3.2 mM EDTA pH 8.0.
EV-Elution B: to volume with PBS with 60 mM NaCl, 45 μM sodium citrate.
After mixing EV-Elution buffer A and B (1× elution solution), 8 μl/ml KH2PO4 were added to the 1× solution before proceeding with EV elution.
Microvesicle Flow Cytometry Analysis
Vesicles from differential ultracentrifugation or from NBI were diluted in 0.22 μm filtered PBS. The background signal was set up based on the acquisition of the filtered PBS, and the light scattering threshold was corrected to allow an acquisition having an event rate of events per second.
Light scattering detection was set in a logarithmic scale, the voltages assigned for the Forward Scattering and the Side Scattering were 300 and 310 V, respectively, and the threshold was set at 200 for both signals. The acquisition was performed at a low flow rate and the samples were carefully diluted to avoid swarm effect and coincidence of events. Standard 1 and 10 μm polystyrene beads (Invitrogen) were used to set the gates for microvesicles. When possible 10,000 events were counted for the analysis of each sample, on the basis of a time acquisition, at least 1 minute acquisition was recorded. Sample acquisition was performed with a FACS Canto flow cytometer (BD Biosciences) and data were analysed using the BD Diva (BD Biosciences) software.
TRPS (Tunable Resistive Pulse Sensing)
EV size and concentration were characterized by TRPS using the qNano (IZON Science) tool. An average of 500 particles were counted for each sample, unless for 6-well plates experiments (
Transmission Electron Microscopy (TEM)
Vesicles were visualized using a transmission electron microscope (TEM). Briefly, a 5 μl aliquot for each EV sample, fixed in elution buffer with 2.5% formaldehyde, was placed on a 300-square mesh grid in copper and nickel coated with a thin carbon film. Grids were then negatively labeled with a 1% uranyl acetate buffer at pH 4.5, and observed using a 100 kV TEM FEI Tecnai G2 Spirit microscope, equipped with an Olympus Morada camera (magnifications used: 20500× and 87000×).
Western blotting analyses were subsequently performed using anti-CD63 antibodies (Abcam, ab193349), anti-Flotillin-1 (BD Biosciences, 610821), and anti-Alix (Cell Signaling Technology, #2171).
Competitive Assay
EV elution step was tested by a competitive assay in which 30 μg/ml of protein extract from DH5a E. coli and 15 μg/ml of recombinant proteins, taggate with histidine, purified (T7 RNA pol, 110 kDa; HuR, 36 kDa; YTH, 23 kDa) were added to 10 ml medium containing EV derived from U87-MG cells. Briefly, DH5a cells were kept in culture in LB medium until an OD600 of 0.5 was reached and were collected by centrifugation at 6000 rcf for 5 min. The pellet was re-suspended in 3 mL DMEM medium+1 μg/ml lysozyme and sonicated at 4° C. in a thermostatic bath for 7 cycles (40 ultrasound amplitude, 7 sec on, 10 sec off). The lysate was clarified by centrifugation at 13,000 rcf for 20 min and then filtered with a 0.2 μm disposable filter membrane before being added to the EV-containing medium. The recombinant protein tagged with histidine T7 RNA polymerase was kindly provided by Dr. S. Mansy's lab (CIBIO, University of Trento); the recombinant proteins HuR (D'Agostino et al., PLoS One, 2013 Aug. 12; 8 (8): e72426) and YTH (Xu et al., J Biol Chem. 2015 Oct. 9; 290: 24902-13) were produced and purified as described in the references.
NBI was performed following incubation times and reagents already described, except for elution gradient solutions indicated in
The number and size of the recovered particles were analysed by TRPS, respecting the sample heterogeneity, using NP800, NP400, and NP200 nanopores.
EV Isolation from Gram-Negative Bacteria
DH5α E. Coli cells were cultured in whole LB medium until OD600 reached 0.7. The cells were pelleted at 4,000 rcf for 15 min and the supernatant was collected to be processed according to NBI protocol. The particles were counted through the qNANO instrument using the NP150 and NP200 nanopores.
Liposome Preparation
The liposome lipid composition, having a lipid composition similar to that of eukaryotic vesicles, is: 20% phosphatidylcholine moles, 10% phosphatidylethanolamine moles, 15% oleophosphatidylserine moles, 15% sphingomyelin moles, 40% cholesterol (Llorente et al. Biochim, Biophys, Acta 2013; 1831: 1302-9; Haraszti et al., J. Extracell, Vesicles 2016; 5: 32570) moles. Lipid films were created removing the organic solvent (e.g. chloroform) from the lipid solution by means of a rotary evaporator instrument and vacuum drying for at least 1 hour. Lipids, at 1 mg/mL final concentration, were re-suspended in DPBS and vigorously stirred with Vortex to form multilamellar liposomes, which were further exposed to 6 freeze-thaw cycles. The final liposome morphology was obtained by extruding a suspension of multilamellar liposomes using a two-syringe extruder (LiposoFast Basic Unit, Avestin Inc.). Thirty-one steps were performed through 2 stacked polycarbonate filters (Millipore) having pores of different size to obtain vesicles of different size (MacDonald et al. Biochim Biophys, Acta 1991; 1061: 297-303), subsequently verified by photon correlation spectroscopy with a Zeta Sizer instrument (Nano-ZS, Malvern Instruments).
Blood Samples
Whole blood samples from healthy donors were collected at Meyer Children's University Hospital. Plasma samples were collected in commercially available EDTA-treated tubes and then shipped from the hospital biological bank to the research laboratories according to cold chain. Informed consent was obtained by donors before sample analysis.
Plasma was obtained by removing cells after centrifugation for 10 minutes at 2,000 rcf using a refrigerated centrifuge (4° C.) (Eppendorf 5702 R, Milan, Italy). Serum samples were obtained by allowing the blood to coagulate, leaving it undisturbed at room temperature for 30 minutes. Coagulum was removed by centrifuging at 2000 rcf using a refrigerated centrifuge (4° C.) (Eppendorf 5702 R, Milan, Italy). The Complete blood count was analysed with a Sysmex XE-5000 flow cytometer (Sysmex America, Mundelein, Ill.). The analytical procedure was conducted according to the protocol instructions.
Immunodeplection of vesicles purified by NBI was performed using anti-CD235a (Miltenyl Biotec, 130-100-271) and anti-CD41a (Miltenyl Biotec, 130-105-608) biotinylated antibodies, respectively, and streptavidin beads (ThermoFisher, 11205D). The vesicles were analysed with qNano after the precipitation of the beads and normalized on the number of particles in the respective control samples with a quantitative equivalent of biotin (Sigma, B4501).
RNA Extraction
Total RNA was extracted using QIAzol reagent (QIAGEN), following the instructions enclosed with some modifications. Briefly, 100 μl of QIAzol were directly added to the beads before EV elution step, subsequently stirred with Vortex and incubated for 5 minutes at room temperature. Then 20 μl of chloroform were added. After vigorously mixing for 15 seconds, the samples were incubated at room temperature for 3 minutes. Phases were separated by centrifugation at 12,000 rcf for 15 minutes at 4° C., and the aqueous phase was drained out. After adding 1 μl glycogen (20 mg/ml) and 100 μl isopropanol, RNA was precipitated overnight at −80° C. After centrifugation at 12,000 rcf for 10 min, RNA pellets were washed with 75% ethanol, centrifuged as described above and re-suspended in 10 μl of RNase-free water. RNA was quantified using Bioanalyzer RNA 6000 Pico Kit (Agilent Technologies) following the protocol instructions.
Reverse Transcription Reaction and Droplet Digital PCR
Reverse transcription reaction was performed using miRCURY LNA Universal RT microRNA PCR kit, Universal cDNA Synthesis Kit II (Exiqon) following the protocol instructions with the following reaction composition: 2.3 μl 5× reaction buffer, 1.15 μl enzyme mix, 0.5 μl synthetic RNA spike-in and 7.5 μl template total RNA. QX200™ Droplet Digital™ PCR System (BioRad) was used to quantify GAPDH mRNA using EvaGreen chemistry and the following primers: 5′-CAACGAATTTGGCTACAGCA-3′ (SEQ ID No. 1) and 5′-AGGGGTCTACATGGCAACTG-3′ (SEQ ID No. 2).
AlphaScreen Assay
The reactions were performed in 384-Optiplate (Perkin Elmer) in a 20 μl final volume. Assay was optimized in PBS using 15 μg/ml nickel-chelate acceptor beads and 10 μg/ml streptavidin-donor beads with serial antibody dilutions to identify the attachment point. The presence of superficial markers was analysed in dose-response with EV serial dilution, previously characterized by TRPS. EVs were purified by NBI from healthy donor plasma or serum-free tumor cell samples. Fluorescence signal was finally detected by Enspire instrument (Perkin Elmer) after 90 minutes of incubation in the dark at room temperature.
Statistical Analysis Data and number of independent experiments are indicated in the relevant captions of the figures. Anova, t-test, and the Pearson r coefficient were calculated by using GraphPad Prism v5.1 software, and the results were considered statistically significant when P value was <0.05 (*), <0.01 (**), <0.001 (***).
Number | Date | Country | Kind |
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102017000146281 | Dec 2017 | IT | national |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2018/085974 | 12/19/2018 | WO | 00 |