CN110068529B - Characterization method of outer vesicle - Google Patents
Characterization method of outer vesicle Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1486—Counting the particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1493—Particle size
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention provides a characterization method of an outer vesicle, which adopts protein fluorescent dye and cell membrane fluorescent dye to carry out double fluorescent staining, obtains multiple parameters of the outer vesicle through a flow cytometer, and realizes evaluation and characterization; according to the invention, through combining the protein fluorescent dye and the cell membrane fluorescent dye, experimental steps and conditions are optimized, and the method is applied to detecting the outer vesicle in the biological sample by the flow cytometer, so that the novel application of the fluorescent dye is widened, and the double staining based on the protein fluorescent dye and the cell membrane fluorescent dye has a better effect in evaluating the outer vesicle.
Description
Technical Field
The invention relates to the field of biochemistry, mainly relates to a characterization method of an outer vesicle, and particularly relates to a characterization method of a microvesicle or/and an exosome.
Background
Extracellular vesicles (extracellular vesicle, EVs) refer to vesicle-like bodies of bilayer membrane structure, with diameters varying from 30nm to 1000nm, that fall off from the cell membrane or are secreted by the cell. Extracellular vesicles are mainly composed of microvesicles (microvesicles) and exosomes (exosomes). Microvesicles are small vesicles that shed from the cell membrane after activation, injury or apoptosis of cells, and have diameters of about 100nm to 1000nm. Exosomes are released outside the cell in the form of exocrine after fusion of the cell membrane with the intracellular multivesicular bodies (Multivesicular bodies), approximately 30-100 nm in diameter. Almost all types of cells, including cancer cells, release exosomes. As an important mediator of intercellular communication, exosomes mediate the transport and exchange of proteins and genetic material.
The existing detection methods of Extracellular Vesicles (EVs) mainly comprise a Scanning Electron Microscope (SEM), an Atomic Force Microscope (AFM), a dynamic light scattering technology (DLS), a Nanoparticle Tracking Analysis (NTA), a flow cytometer, an enzyme-linked immunoassay (ELISA) and the like, and only physical parameters of biological samples can be characterized by the dynamic light scattering technology and the nanoparticle tracking analysis; the sample pretreatment steps of the scanning electron microscope and the atomic force microscope are complex and have higher requirements on experience of operators; because of the high throughput, short time, simple operation, enzyme-linked immunosorbent assay and flow cytometry have become the methods currently used. The ELISA method is easy to be interfered by other soluble antigens, and the information such as the size, the number and the like of the vesicles cannot be known; the flow cytometer can detect the size and the number of the vesicles, and is theoretically the optimal choice for rapid, high-throughput and multi-parameter detection of the vesicles. However, the nano flow type instrument has high cost, and is not beneficial to application and popularization in hospitals and disease detection centers; the above techniques have not been widely used in the medical field so far.
Therefore, a simple and efficient detection method suitable for conventional flow cytometry and used for representing and evaluating exosomes or/and microvesicles is developed, and the method has wide application prospect and great market value.
Disclosure of Invention
Aiming at the defects of the prior art and the market demands, the invention provides a characterization method of the outer vesicle, which combines protein fluorescent dye and cell membrane fluorescent dye, is applied to detecting exosomes or/and microvesicles in biological samples by a flow cytometer, widens the new application of the fluorescent dye, and has better effect in evaluating the outer vesicle.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a method for characterizing an outer vesicle, wherein the method adopts a protein fluorescent dye and a cell membrane fluorescent dye to perform double fluorescent staining, and multiple parameters of the outer vesicle are detected and acquired through a flow cytometer, so that evaluation and characterization of a biological sample are realized.
In the prior art, the methods for identifying microvesicles and exosomes are nanofluidic and scanning electron microscopy, atomic force microscopy, dynamic light scattering technology, nanoparticle tracking analysis, flow cytometry and enzyme-linked immunoassay, but the methods of nanoparticle tracking analysis and enzyme-linked immunoassay can only characterize physical parameters of a sample; while a scanning electron microscope and an atomic force microscope can represent the biological structure of a sample, the requirements on the sample and operators are high; the nanometer flow instrument is a flow instrument specially developed for the outer vesicle, and the purchase cost of the instrument is high at present; all the above factors severely limit the development and popularization of the external vesicle characterization method. The method adopts double staining of protein fluorescent dye of intracellular general esterase and general cell membrane fluorescent dye, and obtains multiparameter of microvesicles and exosomes by a flow cytometer for evaluating the characteristics of the microvesicles and exosomes.
Preferably, the outer vesicles include exosomes or/and microvesicles.
The method can be used for characterization of exosomes and microvesicles, but 100% separation and purification of exosomes or microvesicles cannot be ensured due to the limitation of the current extraction method, and exosomes cannot be specially removed in the experimental step of extracting microvesicles, so that exosomes can be mixed in the microvesicles extracted in general. However, because of the great difficulty in extracting exosomes, the extract is inevitably mixed with microvesicles in a certain proportion. Thus, in practice, the exosomes and microvesicles are present in a mixture. In the prior art, the outer vesicles in a sample are usually counted firstly, then whether the forms of the outer vesicles are complete or not is observed, finally the antigenicity of the outer vesicles is analyzed and verified by means of Western Blot and the like, and the physical activity and the biological activity of the outer vesicles can be evaluated by combining the three methods; the double staining method can monitor the number, the morphology and the biological activity of the outer vesicles in the sample at the same time, and the detection efficiency is obviously improved. In addition, the characterization difficulty of the exosomes is far greater than that of the microvesicles, the grain size of the exosomes is smaller, and the physical and biochemical characterization is difficult to be carried out under the condition of keeping the biological activity by the common operation method.
Preferably, the protein fluorescent dye comprises carboxyfluorescein diacetate succinimidyl ester CFSE.
Preferably, the cell membrane fluorescent dye comprises a lipophilic carbocyanine dye Dil or/and octadecylrhodamine B chloride salt, preferably a lipophilic carbocyanine dye Dil.
The detection sensitivity of the exosomes and the microvesicles is low in a single staining mode, and the two fluorescent dyes are used in combination, so that the characterization accuracy of the exovesicles is improved, the living detection of the exovesicles is realized, and the integrity of a sample is not damaged.
Preferably, the method comprises the steps of:
(1) Separating and extracting the in vitro vesicles;
(2) Mixing carboxyfluorescein diacetate succinimidyl ester CFSE with outer vesicles, and then incubating and staining in a dark place;
(3) Adding a lipophilic carbocyanine dye Dil into the product after incubation and dyeing in the step (2), and incubating and dyeing in a dark place;
(4) And (3) diluting the product after incubation and dyeing in the step (3), detecting by a flow cytometer, and collecting and analyzing data.
The carboxyfluorescein diacetate succinimidyl ester CFSE has no color and does not show fluorescence, and can enter cells through passive diffusion until esterase in the cells removes acetate groups, so that the carboxyfluorescein succinimidyl ester capable of emitting bright fluorescence is generated, and the succinimidyl ester reacts with amino in the cells to form a stable fluorescent conjugate.
The carbocyanine fluorescent dye has longer lipophilic hydrocarbon chain, has the characteristics of high extinction coefficient, polarity dependence, stable coloring, short excited state life, low cytotoxicity and the like, and can be used for living body dyeing of cell membranes and other fat-soluble biological structures. The fluorescent dye with strong light sensitivity has weak fluorescence before entering the cell membrane, can diffuse on the cell membrane once being combined with the cell membrane, greatly enhances the fluorescence intensity, and can uniformly color the cell membrane of a biological sample at the optimal concentration.
The invention adopts double staining of protein fluorescent dye of general esterase-carboxyfluorescein diacetate succinimidyl ester CFSE and general cell membrane fluorescent dye-lipophilic carbocyanine dye Dil, and obtains multiple parameters of microvesicles and exosomes by a flow cytometry for evaluating the characteristics of the microvesicles and exosomes. The common detection method can only physically count the number and concentration of vesicles, and can only roughly evaluate the activity, impurities and the like of a sample by an observation mode. Compared with the traditional method, the characterization method provided by the invention can provide more accurate information, namely the absolute number of the bioactive vesicles.
Preferably, the working concentration of carboxyfluorescein diacetate succinimidyl ester CFSE is 10-80nM, which can be, for example, 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM or 80nM.
Preferably, the lipophilic carbocyanine dye Dil has a working concentration of 0.5-2nM, which may be, for example, 0.5nM, 1nM, 1.5nM or 2nM.
According to the invention, two different fluorescent dyes are specifically selected and the working concentrations are proportioned to realize full dyeing of the outer vesicle, but excessive background noise is not generated, so that subsequent characterization analysis is facilitated, and the characterization effect is reduced if the working concentrations exceed the range of the fluorescent dye: too high a dye concentration may introduce false positive results, and too low a concentration may significantly reduce the staining efficiency resulting in a deviation of results or no results.
Preferably, the temperature of the incubation in step (2) is from 35℃to 37℃and may be, for example, 35 ℃, 36 ℃, or 37 ℃.
Preferably, the incubation time of step (2) is 0.2-3h, and may be, for example, 0.25h, 0.5h, 0.75h, 1h, 1.25h, 1.5h, 1.75h, 2h, 2.25h, 2.5h, 2.75h or 3h.
Preferably, the incubation conditions of step (3) are from 23℃to 26℃for 10 to 30min or from 35℃to 37℃for 1 to 15min, preferably from 23℃to 26℃for 10 to 30min.
In the invention, the common incubation condition of the lipophilic carbocyanine dye Dil is that the lipophilic carbocyanine dye is incubated for 1-15min at the temperature of 35-37 ℃, but the inventor finds that the lipophilic dye has better dyeing effect on a sample plasma membrane under the incubation condition of incubating for 10-30min at the temperature of 23-26 ℃ in the practical process, and can be matched with a protein fluorescent dye to generate a color reaction which does not interfere with each other.
Preferably, the dilution of step (4) is 10-1000 times, e.g. 10-50 times, 100 times, 200 times, 400 times, 600 times, 800 times or 1000 times.
In the invention, the dyeing conditions of different dyes are combined by matching the types of the dyes, and the dilution factors are matched to ensure that the dyeing is full and uniform. Wherein, the dilution factor is in the range of 10-1000 times, and an operator can adjust according to the sample loading requirement of a specific flow cytometer.
As a preferable technical scheme, the characterization method of the outer vesicle specifically comprises the following steps:
(1) Separating and extracting outer vesicles;
(2) Mixing carboxyfluorescein diacetate succinimidyl ester CFSE with the working concentration of 10-80nM with an outer vesicle, and incubating for 0.2-3h at the temperature of 35-37 ℃ in dark for dyeing;
(3) Adding a lipophilic carbocyanine dye Dil with the working concentration of 0.5-2nM into the product after incubation and dyeing in the step (2), and incubating and dyeing in a dark place at 23-26 ℃ for 10-30min or at 35-37 ℃ for 1-15min;
(4) And (3) diluting the product after incubation and dyeing in the step (3) by 10-1000 times, detecting by a flow cytometer, and collecting and analyzing data.
In the invention, in the long-term scientific research practice process, the inventor develops a novel dual fluorescent staining technical method for evaluating microvesicles and exosomes for improving the high efficiency and simplicity of the extracellular vesicle detection method, preferably two fluorescent dyes with different characteristics, optimizes the proportion and experimental steps, and cooperates and increases the conditions of the steps to evaluate the concentration of the microvesicles and the exosomes, the integrity and the bioactivity of the sample, the particle size range of the extracellular vesicles, the content of proteins, lipids and other impurities in the sample and the like, and has wide application prospect and huge market value.
In a second aspect, the present invention provides the use of a composition comprising carboxyfluorescein diacetate succinimidyl ester CFSE and lipophilic carbocyanine dye Dil for dual fluorescent staining of outer vesicles.
Preferably, the outer vesicles are exosomes or/and microvesicles.
Preferably, the use comprises assessing any one or a combination of at least two of the concentration of the outer vesicle, the integrity and biological activity of the outer vesicle sample, the size range of the extracellular vesicle or the content of impurities in the sample.
Preferably, the impurities include protein polymers and lipid compounds.
The steps of centrifugation, filtration, fluorescent staining and the like in the preparation process of the extracellular vesicle sample can cause the disruption of the extracellular vesicle and the introduction of other impurities, and the generated impurities comprise protein polymers and lipid compounds, so that the impurities can be qualitatively and quantitatively treated by the double staining method.
Preferably, the use comprises characterizing the effective concentration of exosomes or/and microvesicles in a biological sample.
The number and concentration of the vesicles can be counted physically only by the common detection method, and the invention can provide more accurate information, namely the absolute number of the vesicles with biological activity by a double-dyeing mode.
Preferably, the use comprises characterizing the integrity and/or breakage rate of exosomes or/and microvesicles structures in a biological sample.
The invention can provide parameters of the particle size distribution range of the exosomes to evaluate the separation efficiency of the exosomes or the microvesicles, the effectiveness of the extraction method and the purity of the sample.
The carboxyfluorescein diacetate succinimidyl ester and the lipophilic carbocyanine fluorescent dye are used for dyeing phospholipid bilayer cell membrane structures of microvesicles and exosomes and intracellular proteins, and can be used for detecting the bioactivity and evaluating the quality of the cells. The double staining scheme related by the invention can be used for separating and extracting microvesicles and exosomes for living body detection and quality evaluation, long-time multi-parameter monitoring of clinical patients and disease diagnosis; dual staining based on carboxyfluorescein diacetate succinimidyl ester dye can be used to evaluate effective concentration of microvesicles and exosomes, sample integrity and bioactivity, particle size range of extracellular vesicles, content of impurities such as proteins and lipids in sample, etc.
The invention relates to a novel double fluorescent staining technical method for evaluating microvesicles and exosomes. The carboxyfluorescein diacetate succinimidyl ester dye can display fluorescence under the action of intracellular proteins, and the lipophilic carbocyanine fluorescent dye can display fluorescence by effectively combining with phospholipid bilayer structures of cell membranes. The signal of the fluorophore was recorded by flow cytometry for subsequent data analysis and quality assessment. The invention finds that double staining based on carboxyfluorescein diacetate succinimidyl ester dye has better effect in evaluating microvesicles and exosomes.
The carboxyfluorescein diacetate succinimidyl ester and the lipophilic carbocyanine fluorescent dye are respectively used as fluorescent dyes of proteins and cell membranes, are applied to characterization of microvesicles and exosome biological structures, develop new application of the combination of the two fluorescent dyes, and provide new choices for activity evaluation after in vitro extraction of the microvesicles and exosomes. Specifically, the application of double staining in the extraction efficiency of microvesicles and exosomes in the characterization biological sample; the use of double staining to characterize the integrity/breakage rate of microvesicle and exosome structures in biological samples; the use of double staining to characterize microvesicle and exosome content in biological samples; use of double staining for characterizing the size distribution interval of microvesicles and exosomes in biological samples. The traditional method cannot reflect the biological activity of the vesicle through one counting experiment, but can roughly evaluate the biological activity through antigens on a plasma membrane and vesicle morphology under an electron microscope. The characterization method of the invention can evaluate the bioactivity of the vesicle by adopting a flow cytometry method, realizing the counting of the vesicle on one hand and realizing the data statistics of the vesicle plasma membrane and the protein wrapped in the vesicle plasma membrane by adopting a double-dyeing method on the other hand.
Compared with the prior art, the invention has the following advantages:
the double dyeing of the carboxyfluorescein diacetate succinimidyl ester and the lipophilic carbocyanine fluorescent dye provided by the invention is applied to the characterization and evaluation of exosomes or/and microvesicles, the method is simple and efficient, can detect multiple parameters simultaneously, realizes accurate counting and evaluation of the biological activity of a sample, and has the advantages of small dosage, low toxicity, broad spectrum, low price and the like.
Detailed Description
In order to further describe the technical means adopted by the present invention and the effects thereof, the following describes the technical scheme of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
Example 1
Exosome-free serum cultures 1X 10 6 Hela cells at 37℃and 5% CO 2 After 6 hours, the culture supernatant was collected and used for the separation and extraction of microvesicles and exosomes.
And (3) performing ultracentrifugation at 4 ℃ for 2h at 100,000g, performing ultracentrifugation twice, and re-dissolving in PBS solution to obtain the extracted exosomes.
Carboxyfluorescein diacetate succinimidyl ester (eBioscience) with a final concentration of 25nM TM CFSE, cat No.: 65-0850-84, thermo filter) solution and exosomes, incubating for 1.5 hours in an incubator at 37 ℃ in the absence of light;
then, a lipophilic carbocyanine fluorescent dye ('DiI'; diIC18 (3), cat# D282, thermo fisher) solution and exosomes were incubated at a final concentration of 1nM for 10min at 37℃in the absence of light or for 20min at ambient (24 ℃) in the absence of light.
The stained samples were diluted 100-fold and subsequently subjected to flow data collection and analysis.
The exosomes separated by ultrafiltration technology are evaluated based on the method, and the concentration of the extracted nano-sized particles is found to be 3.2-7.6X10 8 /mL; wherein the content of double-stained extracellular vesicles is 53.1% -63.3%; the protein polymer content in the solution is 10.5% -14.1%; the content of cell membrane fragments is 10.2% -17.1%, and the content of other impurities is 5.5-7.8%.
Example 2
Exosome-free serum cultures 1X 10 6 Hela cells at 37℃and 5% CO 2 After 6 hours, the culture supernatant was collected and used for the separation and extraction of microvesicles and exosomes.
And (3) performing ultracentrifugation at 4 ℃ for 2h at 100,000g, performing ultracentrifugation twice, and re-dissolving in PBS solution to obtain the extracted exosomes.
Carboxyfluorescein diacetate succinimidyl ester (eBioscience) with a final concentration of 80nM TM CFSE, cat No.: 65-0850-84, thermo fisher) solution and exosomes, incubating for 0.2h in an incubator at 37 ℃ in the absence of light;
then, a lipophilic carbocyanine fluorescent dye ('DiI'; diIC18 (3), cat# D282, thermo fisher) solution and exosomes were incubated at a final concentration of 2nM for 1min at 37℃in the absence of light or for 10min at ambient (24 ℃) in the absence of light.
The stained samples were diluted 1000-fold and subsequently subjected to flow data collection and analysis.
The exosomes separated by ultrafiltration technology are evaluated based on the method, and the concentration of the extracted nano-sized particles is found to be 5.6-7.6X10 8 /mL; wherein the content of double-stained extracellular vesicles is 55.2% -61.9.3%; the protein polymer content in the solution is 11.2% -15.1%; the content of cell membrane fragments is 11.7% -16.3%, and the content of other impurities is 4.9-8.6%.
Example 3
Exosome-free serum cultures 1X 10 6 Hela cells at 37℃and 5% CO 2 After 6 hours, the culture supernatant was collected and used for the separation and extraction of microvesicles and exosomes.
And (3) performing ultracentrifugation at 4 ℃ for 2h at 100,000g, performing ultracentrifugation twice, and re-dissolving in PBS solution to obtain the extracted exosomes.
Carboxyfluorescein diacetate succinimidyl ester (eBioscience) with a final concentration of 10nM TM CFSE, cat No.: 65-0850-84, thermo filter) solution and exosomes, incubating for 3 hours in an incubator at 37 ℃ in the absence of light;
then, a lipophilic carbocyanine fluorescent dye ('DiI'; diIC18 (3), cat# D282, thermo fisher) solution and exosomes were incubated at a final concentration of 0.5nM for 15min at 37℃in the absence of light or for 30min at ambient (24 ℃) in the absence of light.
The stained samples were diluted 10-fold and subsequently subjected to flow data collection and analysis.
The exosomes separated by ultrafiltration technology are evaluated based on the method, and the concentration of the extracted nano-sized particles is found to be 1.2-8.6x10 8 /mL; wherein the content of double-stained extracellular vesicles is 48.1% -63.3%; protein polymer content in solution 10.8% -17.2%; the content of cell membrane fragments is 9.3% -15.7%, and the content of other impurities is 5.2-8.9%.
Example 4 characterization of extraction efficiency applied to Hela microvesicles and exosomes
Carboxyfluorescein diacetate succinimidyl ester (eBioscience) TM CFSE, cat No.: 65-0850-84, thermo cleaner) is prepared into required concentration by dimethyl sulfoxide, filtered and sterilized, and the solution is preserved in dark for a short period (within half a year) at-20 ℃.
Lipophilic carbocyanine fluorescent dyes ('DiI'; diIC18 (3), cat# D282, thermo cleaner) were formulated with absolute ethanol/dimethylformamide/dimethylsulfoxide to the desired concentration, and the solution was stored in the dark for a short period of time (within half a year) at-20 ℃.
Carboxyfluorescein diacetate succinimidyl ester (CFSE) solution with final concentration of 10nM and exosomes were incubated in an incubator at 37℃for 2h protected from light;
then, the lipophilic carbocyanine fluorescent dye solution with a final concentration of 0.5nM and the above-mentioned CFSE-stained exosomes were incubated for 15min at 37℃or 30min at normal temperature (23 ℃ -26 ℃) in the absence of light.
The stained samples were diluted 100-fold for cell flow data collection and analysis.
The blank of this example was an equal volume of PBS solution and the other treatments included carboxyfluorescein diacetate succinimidyl ester single stain, lipophilic carbocyanine dye single stain, and double stain, where "++" indicates CFSE and Dil were used in double stain at the same concentration as the single stain, i.e., 10nM dil+1nM CFSE.
The results of the experimental data are shown in table 1:
TABLE 1 characterization of extracellular vesicle samples
As shown in table 1, the PBS buffer does not have any staining effect on the sample, and impurity lipid is also present in the Dil single staining result while the outer vesicle is included, the staining ratio of the lipid in the Dil stained sample is a+b=56.91%, but the method cannot distinguish the outer vesicle from the impurity lipid, and cannot detect the presence and the content of the impurity protein polymer; the CFSE single staining result comprises the outer vesicle and the impurity protein polymer, the staining proportion of the protein in the CFSE stained sample is 82.16%, but the method cannot distinguish the outer vesicle from the impurity protein polymer and cannot detect the existence and the content of the impurity lipid; while double staining with cfse+dil can stain proteins and lipids, samples with both colors are outer vesicles, while the fraction with only a single stain is the corresponding impurity lipid or impurity protein polymer. The physical and biological activities of the external vesicles can be evaluated by using the double staining method of the invention and a common flow cytometer. As can be seen from the description in Table 1, the ratio of extracellular vesicles in the sample solution was 63.28%, and the sample also contained 14.12% extracellular protein polymer, 17.13% lipid and 5.57% other impurities. Compared with single staining, the double staining method can more accurately evaluate the content of extracellular vesicles and impurity components and content in the solution.
Example 5 particle size analysis of extracellular vesicles from lung cancer
100nm, 200nm, 300nm and 500nm polystyrene microspheres (FluoSpheres) were used in this example TM Size Kit #2, cat: f8888, thermo Fisher), diluted 100,000-fold with PBS solution for flow analysis.
The final concentration is 10nM, and the carboxyfluorescein diacetate succinimidyl ester solution and exosome are incubated for 2h in a 37 ℃ incubator in the absence of light; then, the lipophilic carbocyanine fluorescent dye solution and exosomes were incubated at a final concentration of 1nM for 30min at ambient temperature (23 ℃ -26 ℃) protected from light. The experimental results are shown in Table 2.
TABLE 2 particle size and content of extracellular vesicles from lung cancer
Dil single staining is a cell lipid impurity, CFSE single staining is impurity protein, and the areas where neither dye is stained are other impurities. From Table 2, it is clear that the particle size of the lung cancer extracellular vesicles is more distributed in 200-500nm and the proportion in the 100nm interval is smaller according to the data analysis under the parameter setting of the violet (405 nm) side scattered light (VSSC) of the flow cytometer. In addition, the sample has very little extracellular protein, but the content of lipid and other impurities is higher, especially in the range of 100-300 nm. The traditional method cannot reflect the biological activity of the vesicle through one counting experiment, but can roughly evaluate the biological activity through antigens on a plasma membrane and vesicle morphology under an electron microscope. The method can evaluate the bioactivity of the vesicle by adopting a flow cytometry method, on one hand, counting the vesicle, and on the other hand, adopting a double-dyeing method to realize the data statistics of the vesicle plasma membrane and the protein wrapped in the vesicle plasma membrane.
In summary, the invention provides a method for characterizing exosomes or/and microvesicles, which combines carboxyfluorescein diacetate succinimidyl ester with carbocyanine fluorescent dye, optimizes experimental steps and conditions, is applied to flow cytometry to detect exosomes or/and microvesicles in biological samples, widens new application of fluorescent dye, and has better effect in evaluating microvesicles and exosomes based on double staining of carboxyfluorescein diacetate succinimidyl ester dye.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (13)
1. The characterization method of the outer vesicle is characterized in that the method adopts protein fluorescent dye and cell membrane fluorescent dye to carry out double fluorescent staining, and obtains multiple parameters of the outer vesicle through a flow cytometer to realize evaluation and characterization;
the protein fluorescent dye comprises carboxyfluorescein diacetate succinimidyl ester CFSE;
the cell membrane fluorescent dye comprises lipophilic carbocyanine dye Dil or/and octadecyl rhodamine B chloride salt;
the working concentration of the carboxyfluorescein diacetate succinimidyl ester CFSE is 10-80nM;
the working concentration of the lipophilic carbocyanine dye Dil is 0.5-2nM.
2. The characterization method of claim 1 wherein the exovesicles include exosomes or/and microvesicles.
3. The characterization method of claim 1 wherein the cell membrane fluorescent dye is a lipophilic carbocyanine dye Dil.
4. A method according to any one of claims 1-3, characterized in that the method comprises the steps of:
(1) Separating and extracting outer vesicles;
(2) Mixing carboxyfluorescein diacetate succinimidyl ester CFSE with outer vesicles, and then incubating and staining in a dark place;
(3) Adding a lipophilic carbocyanine dye Dil into the product after incubation and dyeing in the step (2), and incubating and dyeing in a dark place;
(4) And (3) diluting the product after incubation and dyeing in the step (3), detecting by a flow cytometer, and collecting and analyzing data.
5. The method of claim 4, wherein the incubation in step (2) is at a temperature of 35 ℃ to 37 ℃;
the incubation time in the step (2) is 0.2-3h.
6. The method according to claim 4, wherein the incubation conditions of step (3) are 23 ℃ to 26 ℃ for 10 to 30min or 35 ℃ to 37 ℃ for 1 to 15min.
7. The method of claim 6, wherein the incubation conditions of step (3) are 23 ℃ to 26 ℃ for 10 to 30min.
8. The method of claim 4, wherein the dilution of step (4) is 10-1000 fold.
9. The method according to claim 4, characterized in that it comprises the following steps:
(1) Separating and extracting outer vesicles;
(2) Mixing carboxyfluorescein diacetate succinimidyl ester CFSE with the working concentration of 10-80nM with an outer vesicle, and incubating for 0.2-3h at the temperature of 35-37 ℃ in dark for dyeing;
(3) Adding a lipophilic carbocyanine dye Dil with the working concentration of 0.5-2nM into the product after incubation and dyeing in the step (2), and incubating and dyeing in a dark place at 23-26 ℃ for 10-30min or at 35-37 ℃ for 1-15min;
(4) And (3) diluting the product after incubation and dyeing in the step (3) by 10-1000 times, detecting by a flow cytometer, and collecting and analyzing data.
10. Use of a composition comprising carboxyfluorescein diacetate succinimidyl ester CFSE and lipophilic carbocyanine dye Dil for dual fluorescent staining of outer vesicles;
the working concentration of the carboxyfluorescein diacetate succinimidyl ester CFSE is 10-80nM;
the working concentration of the lipophilic carbocyanine dye Dil is 0.5-2nM.
11. The use according to claim 10, wherein the exovesicles are exosomes or/and microvesicles.
12. The use of claim 11, comprising assessing any one or a combination of at least two of the effective concentration of the outer vesicle, the integrity and bioactivity of the outer vesicle sample, the size range of the extracellular vesicle, or the impurity content in the sample;
the impurities include protein polymers and lipid compounds.
13. The use according to claim 11, characterized in that it comprises characterizing the integrity rate or/and the breakage rate of exosomes or/and microvesicles structures in a biological sample.
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