A method for identifying an extracellular vesicle

Abstract

The application discloses a method for identifying extracellular vesicles, and relates to the technical field of identification of extracellular vesicles. The method comprises the following steps in sequence: dropping a polylysine solution on a glass slide, carrying out coating, air-drying, then adding an extracellular vesicle solution, incubating at room temperature, then adding a fluorescein-5-maleimide solution, incubating at room temperature in the dark, and then carrying out fluorescence identification under a confocal microscope. The method realizes rapid detection of the vesicles by using fluorescein-5-maleimide for marking detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of identification of extracellular vesicles, and particularly relates to an identification method of extracellular vesicles. BACKGROUND

[0002] Extracellular vesicles (EVs) refer to vesicle-like small bodies with double-membrane structure which are shed from cell membranes or secreted by cells, and the diameters thereof are from 30nm to 10000nm. Extracellular vesicles are mainly composed of microvesicles (MVs) and exosomes (Exos). The microvesicles are small vesicles which are shed from cell membranes after cell activation, injury or apoptosis, and the diameters thereof are about 150nm-10000nm. The exosomes are released to the extracellular space in the form of excretion after the fusion of intracellular multivesicular bodies with cell membranes, and the diameters thereof are about 30nm-150nm. Extracellular vesicles widely exist in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk), carry a variety of proteins, lipids, DNA, mRNA, miRNA and the like related to cell sources, and are involved in cell communication, cell migration, angiogenesis and immune regulation. The level of extracellular vesicles is found to be increased in diabetes, cardiovascular disease, AIDS, chronic inflammatory disease and cancer, and they are likely to become diagnostic markers of these diseases. Therefore, it is particularly important to accurately separate and identify extracellular vesicles.

[0003] At present, the identification methods of extracellular vesicles (EVs) mainly include transmission electron microscopy, flow cytometry, enzyme-linked immunosorbent assay, Western blotting, dynamic light scattering technology and nanoparticle tracking analysis technology. The above methods have the disadvantages of complex operation, poor repeatability and unsuitability for detecting marker proteins in biological fluids. SUMMARY

[0004] In order to solve the above technical problems, the purpose of the present application is to provide an identification method of extracellular vesicles, so as to solve the problems of complex operation and poor repeatability of the existing identification methods.

[0005] The technical scheme for solving the above technical problems is as follows: an identification method of extracellular vesicles is provided, which comprises the following steps in sequence:

[0006] (1) a polylysine solution is dropped on a glass slide, coated for 30-50min, air-dried, and then an extracellular vesicle solution is added and incubated at room temperature for 25-35min;

[0007] (2) drop the fluorescein-5-maleimide solution, incubate at room temperature for 25-35 min in the dark, and then perform fluorescence identification under a confocal microscope.

[0008] Based on the technical solution, the application can be further improved as follows:

[0009] Further, in step (1), the concentration of the polylysine solution is 90-110 µg / mL.

[0010] Further, in step (1), the concentration of the polylysine solution is 100 µg / mL.

[0011] Further, in step (1), the concentration of the extracellular vesicle solution is 0.8-1.2 µg / µL.

[0012] Further, in step (1), the concentration of the extracellular vesicle solution is 1 µg / µL.

[0013] Further, in step (2), the concentration of the fluorescein-5-maleimide solution is 1.8-2.2 µmol / L.

[0014] Further, in step (2), the concentration of the fluorescein-5-maleimide solution is 2 µmol / L.

[0015] Further, in steps (1)-(2), the volume ratio of the polylysine solution, the extracellular vesicle solution, and the fluorescein-5-maleimide solution is 1:1:0.8-1.2.

[0016] Further, in steps (1)-(2), the volume ratio of the polylysine solution, the extracellular vesicle solution, and the fluorescein-5-maleimide solution is 1:1:1.

[0017] The application also provides the use of the above method in the identification of extracellular vesicles.

[0018] The application has the following advantages:

[0019] The application realizes the adhesion and fluorescent labeling of extracellular vesicles, develops a specific labeling technology for extracellular vesicle markers, and can accurately observe extracellular vesicles and their markers using a confocal microscope. The detection can be completed within 3 hours, realizing fast detection of vesicles. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 An operation flowchart for the co-labeling of extracellular vesicles, F5M, and CD63;

[0021] Figure 2 A Fourier infrared spectrum of extracellular vesicles;

[0022] Figure 3 Fluorescence graph for extracellular vesicle and CD63 binding;

[0023] Figure 4 Fluorescence graph for extracellular vesicle and F5M binding;

[0024] Figure 5 Fluorescence graph for extracellular vesicle F5M and CD63 binding. DETAILED DESCRIPTION

[0025] The principles and features of the present application are described below with reference to the accompanying drawings, and the examples are used only to explain the present application and are not intended to limit the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not mentioned by the manufacturer, and are all conventional products that can be purchased on the market.

[0026] Example 1:

[0027] A method for identifying extracellular vesicles, comprising the following steps in sequence:

[0028] 100 μL of a solution of polylysine (PLL) with a concentration of 100 μg / mL was dropped on a glass slide, coated for 40 min, air-dried, and PLL was cross-linked with the glass slide so as to adhere extracellular vesicles later. Then 100 μL of the extracellular vesicle solution obtained in step (1) (with a concentration of 1 μg / μL) was added, incubated at room temperature for 30 min, and extracellular vesicles were cross-linked with PLL. Then 100 μL of a solution of fluorescein-5-maleimide (F5M) (with a concentration of 2 μmol / L) was added, incubated at room temperature in the dark for 30 min, and F5M was combined with extracellular vesicles. Fluorescence identification was performed under a confocal microscope. Figure 1 The first three steps in the above method

[0029] Example 2:

[0030] A method for identifying extracellular vesicles, comprising the following steps in sequence:

[0031] 100 μL of a solution of polylysine (PLL) with a concentration of 90 μg / mL was dropped on a glass slide, coated for 30 min, air-dried, and PLL was cross-linked with the glass slide so as to adhere extracellular vesicles later. Then 100 μL of the extracellular vesicle solution obtained in step (1) (with a concentration of 0.8 μg / μL) was added, incubated at room temperature for 25 min, and extracellular vesicles were cross-linked with PLL. Then 80 μL of a solution of fluorescein-5-maleimide (F5M) (with a concentration of 1.8 μmol / L) was added, incubated at room temperature in the dark for 25 min, and F5M was combined with extracellular vesicles. Fluorescence identification was performed under a confocal microscope.

[0032] Example 3:

[0033] A method for identifying extracellular vesicles, comprising the following steps in sequence:

[0034] 100 μL of a solution of polylysine (PLL) at a concentration of 110 μg / mL was dropped on a glass slide, coated for 50 min, air-dried, and cross-linked with the glass slide to facilitate the subsequent adhesion of extracellular vesicles. Then 100 μL of the extracellular vesicle solution obtained in step (1) (at a concentration of 1.2 μg / μL) was added, incubated at room temperature for 35 min to cross-link the extracellular vesicles with the PLL, and then 120 μL of a solution of fluorescein-5-maleimide (F5M) at a concentration of 2.2 μmol / L was added, incubated at room temperature in the dark for 35 min to allow the F5M to bind to the extracellular vesicles, and then fluorescence identification was performed under a confocal microscope.

[0035] Test Example

[0036] I. Vesicle Labeling-Infrared Detection

[0037] (1) Perform a ligation reaction, and add the following amounts of reagents for each group:

[0038]

[0039] The concentration of the extracellular vesicles was 3 mg / mL, and the concentration of the F5M stock solution was 3 μmol / L, and the final concentration was 2 μmol / L.

[0040] (2) Perform the reaction according to the above table, and vortex the suspension evenly during the reaction. Incubate the suspension at room temperature in the dark for 30 min.

[0041] (3) Mix 90 uL of the sample directly with 0.2 g of KBr powder, dry in an oven at 60°C, press the tablet in a tablet press mold, and detect using a Fourier transform infrared spectrometer. The FTIR spectral wave number range is 4000-650 cm -1 , and the average resolution is 4 cm -1 .

[0042] (4) Process and analyze the data using GraphPad software, and the results are shown in Figure 2 (the concentration decreases from bottom to top in the vertical coordinate at 3000-4000).

[0043] As can be seen from Figure 2 , FTIR spectral analysis confirms the ligation of extracellular vesicles and maleimide, and there are differences in the ligation of extracellular vesicles and maleimide at different concentrations. The experiment shows that the thioether bond between maleimide and the thiol group is shown in the spectrum, indicating that F5M is successfully combined with extracellular vesicles, and the higher the concentration of the vesicles, the higher the binding efficiency, but it reaches saturation at 0.4 μg / μL.

[0044] II. Antibody labeling - fluorescent detection (take biomarker CD63 as an example)

[0045] 1. CD63 labeling of extracellular vesicles

[0046] (1) 100 μg / mL PLL (poly-L-lysine) solution was used to coat the glass slide at room temperature for 40 min, so as to crosslink PLL with the glass slide, for the purpose of adhering extracellular vesicles later;

[0047] (2) 100 μL of 1 μg / μL extracellular vesicles was added on the coated glass slide, and incubated at room temperature for 30 min, so as to crosslink extracellular vesicles with PLL;

[0048] (3) 100 μL of CD63 (Anti-CD63 antibody [EPR5702], primary antibody, abcam (ab134045)) diluted at 1:500 was added on the glass slide, and incubated at room temperature in the dark for 30 min, so as to bind CD63 with EVs;

[0049] (4) 50 μL of secondary antibody (Alexa Fluor 555 labeled donkey anti-rabbit IgG (H+L), Biyun Tian (A0453)) with fluorescent labeling was added, diluted at 1:1000, and incubated at room temperature in the dark for 30 min;

[0050] (5) The fluorescence of exosomes was observed under a confocal microscope with a 63x objective lens, using 555 nm excitation light. The results are shown in Figure 3 .

[0051] 2. F5M labeling of extracellular vesicles

[0052] (1) 100 μg / mL PLL (poly-L-lysine) solution was used to coat the glass slide at room temperature for 40 min, so as to crosslink PLL with the glass slide, for the purpose of adhering extracellular vesicles later;

[0053] (2) 100 μL of 1 μg / μL extracellular vesicles was added on the coated glass slide, and incubated at room temperature for 30 min, so as to crosslink extracellular vesicles with PLL;

[0054] (3) 100 μL of fluorescein-5-maleimide (F5M) 2 μM was added, and incubated at room temperature in the dark for 30 min, so as to bind F5M with extracellular vesicles;

[0055] (4) The fluorescence of exosomes was observed under a confocal microscope with a 63x objective lens, using 494 nm excitation light. The results are shown in Figure 4 .

[0056] 3. F5M and CD63 co-labeling (operation process is shown in Figure 1 )

[0057] (1) 100 μg / mL PLL (poly-L-lysine) solution coated glass slides at room temperature for 40 min, so that the PLL cross-linking with the glass, so as to adhere to the extracellular vesicles later.

[0058] (2) extracellular vesicles 1 μg / μL 100 μL drop on coated glass slides, incubated at room temperature for 30 min, so that the extracellular vesicles cross-linking with PLL.

[0059] (3) fluorescein-5-maleimide (F5M) 2 μM 100 μL drop, incubated at room temperature for 30 min in the dark, so that the F5M combined with extracellular vesicles.

[0060] (4) extracellular vesicle-specific markers combined with F5M-EVs (fluorescein-5-maleimide-extracellular vesicles), the experiment selected CD63, CD63 diluted 1:500 after 100 μL drop on the glass, incubated at room temperature for 30 min in the dark, so that the CD63 combined with F5M-EVs.

[0061] (5) 50 μL with fluorescently labeled secondary antibody Alexa Fluor 555-labeled Donkey Anti-Rabbit IgG (H+L) was added, diluted 1:1000, incubated at room temperature for 30 min in the dark, so that the secondary antibody combined with CD63 in F5M-EVs-CD63 specific.

[0062] (6) the excitation wavelength of F5M is 494 nm, the excitation wavelength of the secondary antibody is 555 nm, under the 63x objective lens of confocal microscope, different excitation light is selected to observe the fluorescence of exosomes.

[0063] Results are shown in Figure 5 (scale 10 μm, CD63 is red fluorescence, F5M is green fluorescence).

[0064] From Figures 3-5 It can be seen that CD63 and F5M can be combined with extracellular vesicles, and the number is large. When the extracellular vesicles are combined with F5M first, and then combined with CD63, the fluorescence results show that both markers are combined with extracellular vesicles, and can jointly label extracellular vesicles.

[0065] The above only describes the preferred embodiments of the present application, and does not limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for identifying an extracellular vesicle, characterized by, Sequentially comprising the following steps: (1) drop polylysine solution on the slide, coat for 30-50 min, air dry, then add extracellular vesicle solution, incubate at room temperature for 25-35 min; (2) add fluorescein-5-maleimide solution, incubate at room temperature in the dark for 25-35 min, then perform fluorescence identification under a confocal microscope. 2.The method of identifying extracellular vesicles according to claim 1, characterized in that, In step (1), the concentration of the polylysine solution is 90-110 μg / mL. 3.The method of identifying extracellular vesicles according to claim 1, characterized in that, In step (1), the concentration of the polylysine solution is 100 μg / mL. 4.The method of identifying extracellular vesicles according to claim 1, characterized in that, In step (1), the concentration of the extracellular vesicle solution is 0.8-1.2 μg / μL. 5.The method of identifying extracellular vesicles according to claim 1, characterized in that, In step (1), the concentration of the extracellular vesicle solution is 1 μg / μL. 6.The method of identifying extracellular vesicles according to claim 1, characterized in that, In step (2), the concentration of the fluorescein-5-maleimide solution is 1.8-2.2 μmol / L. 7.The method of identifying extracellular vesicles according to claim 1, characterized in that, In step (2), the concentration of the fluorescein-5-maleimide solution is 2 μmol / L. 8.The method of identifying extracellular vesicles according to claim 1, characterized in that, In steps (1)-(2), the volume ratio of the polylysine solution, the extracellular vesicle solution and the fluorescein-5-maleimide solution is 1:1:0.8-1.

2. 9.The method of identifying extracellular vesicles according to claim 1, characterized in that, In steps (1)-(2), the volume ratio of the polylysine solution, the extracellular vesicle solution and the fluorescein-5-maleimide solution is 1:1:

1.

10. Use of the method for identifying extracellular vesicles according to any one of claims 1-9 in the identification of extracellular vesicles.

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