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Characterization method of extracellular vesicle

A vesicle and characterization technology, applied in the field of biochemistry, can solve the problems of unfavorable application and promotion of hospitals and disease detection centers, high cost of nanometer flow meters, etc., and achieve the effect of improving the accuracy of characterization

Active Publication Date: 2019-07-30
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high cost of nanometer flow is not conducive to the application and promotion in hospitals and disease detection centers; so far, the above technologies have not been widely used in the medical field

Method used

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  • Characterization method of extracellular vesicle
  • Characterization method of extracellular vesicle
  • Characterization method of extracellular vesicle

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Serum culture without exosomes 1 × 10 6 HeLa cells at 37 °C and 5% CO 2 Under the conditions, the medium supernatant was collected after 6 hours for the separation and extraction of microvesicles and exosomes.

[0054] 4°C, 100,000g ultracentrifugation for 2 hours, after two ultrahigh-speed centrifugation, and then redissolved in PBS solution to obtain the extracted exosomes.

[0055] Carboxyfluorescein diacetate succinimidyl ester (eBioscience TM CFSE, product number: 65-0850-84, Thermo fisher) solution and exosomes, co-incubated in a 37°C incubator in the dark for 1.5h;

[0056] Then, lipophilic carbocyanine fluorescent dye ('DiI'; DiIC18(3), product number: D282, Thermo fisher) solution and exosomes with a final concentration of 1 nM were co-incubated at 37°C for 10min in the dark or at room temperature ( 24°C) and incubated in the dark for 20min.

[0057] The stained samples were diluted 100-fold for subsequent flow cytometric data collection and analysis.

[0...

Embodiment 2

[0060] Serum culture without exosomes 1 × 10 6 HeLa cells at 37 °C and 5% CO 2 Under the conditions, the medium supernatant was collected after 6 hours for the separation and extraction of microvesicles and exosomes.

[0061] 4°C, 100,000g ultracentrifugation for 2 hours, after two ultrahigh-speed centrifugation, and then redissolved in PBS solution to obtain the extracted exosomes.

[0062] Carboxyfluorescein diacetate succinimidyl ester (eBioscience TM CFSE, product number: 65-0850-84, Thermo fisher) solution and exosomes, co-incubated in a 37°C incubator in the dark for 0.2h;

[0063] Then, lipophilic carbocyanine fluorescent dye ('DiI'; DiIC18(3), product number: D282, Thermo fisher) solution and exosomes with a final concentration of 2nM were co-incubated at 37°C for 1min in the dark or at room temperature ( 24°C) and incubated in the dark for 10 minutes.

[0064] The stained samples were diluted 1000 times for subsequent flow cytometric data collection and analysis. ...

Embodiment 3

[0067] Serum culture without exosomes 1 × 10 6 HeLa cells at 37 °C and 5% CO 2 Under the conditions, the medium supernatant was collected after 6 hours for the separation and extraction of microvesicles and exosomes.

[0068] 4°C, 100,000g ultracentrifugation for 2 hours, after two ultrahigh-speed centrifugation, and then redissolved in PBS solution to obtain the extracted exosomes.

[0069] Carboxyfluorescein diacetate succinimidyl ester (eBioscience TM CFSE, product number: 65-0850-84, Thermo fisher) solution and exosomes, co-incubated in a 37°C incubator in the dark for 3h;

[0070] Then, lipophilic carbocyanine fluorescent dye ('DiI'; DiIC18(3), product number: D282, Thermo fisher) solution and exosomes with a final concentration of 0.5nM were co-incubated at 37°C for 15min in the dark or at room temperature. (24°C) and incubate for 30 min in the dark.

[0071] The stained samples were diluted 10 times for subsequent flow cytometric data collection and analysis.

[00...

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Abstract

The invention provides a characterization method of extracellular vesicle. The method comprises the following steps; performing double fluorescence staining by adopting protein fluorochrome and cytomembrane fluorochrome, and acquiring multiple parameters of the extracellular vesicle through a flow cytometry, thereby realizing assessment characterization. By combining the protein fluorochrome and the cytomembrane fluorochrome and optimizing experimental steps and conditions, the characterization method is applied to detecting the extracellular vesicle in the biological sample by using the flowcytometry, the new application of the fluorochrome is broadened, and the double staining based on the protein fluorochrome and the cytomembrane fluorochrome has good effect for assessing the extracellular vesicle.

Description

technical field [0001] The present invention relates to the field of biochemistry, and mainly relates to a method for characterizing exovesicles, in particular to a method for characterizing microvesicles or / and exosomes. Background technique [0002] Extracellular vesicles (EVs) refer to vesicle-like bodies with a double-membrane structure shed from the cell membrane or secreted by cells, with diameters ranging from 30 nm to 1000 nm. Extracellular vesicles are mainly composed of microvesicles and exosomes. Microvesicles are small vesicles that are shed from the cell membrane after cell activation, injury or apoptosis, with a diameter of about 100nm–1000nm. Exosomes are released outside the cell in the form of exocrine after the fusion of multivesicular bodies (Multivesicular bodies) with the cell membrane, with a diameter of about 30nm-100nm. Almost all types of cells, including cancer cells, release exosomes. As an important medium of intercellular communication, exosom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14G01N21/64
CPCG01N15/1434G01N21/6428G01N2015/1486G01N2015/1493G01N2021/6439
Inventor 杨慧杜静
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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