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Fluorescent labeling method for living organisms having cell membrane structures

A technology of fluorescent labeling and fluorescent markers, applied in the field of fluorescent labeling, can solve the problems of large influence on biological activity, low efficiency, and cumbersome two-step labeling operations

Inactive Publication Date: 2015-07-01
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a fluorescent labeling method for modifying organisms with cell membrane structures with choline analogs containing azide groups, so as to solve the existing problems based on click chemistry. Fluorescent labeling method has a great impact on the technical problems of biological activity
[0007] Further, the object of the present invention is to provide a one-step fluorescent labeling method with simple operation and high efficiency, so as to solve the technical problems of cumbersome operation and low efficiency of the existing two-step labeling method

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  • Fluorescent labeling method for living organisms having cell membrane structures
  • Fluorescent labeling method for living organisms having cell membrane structures
  • Fluorescent labeling method for living organisms having cell membrane structures

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preparation example Construction

[0029] The preparation method of the above-mentioned choline analogue containing azide group can be synthesized by referring to the method disclosed in the literature Analytical chemistry 201385 (10), 5263-5270. For the specific preparation method, please refer to the content of raw materials and reagents in the following examples.

[0030]The above-mentioned choline analogue containing an azide group has an azide group at one end for click chemical reaction; the other end is a choline molecule, which can participate in the lipid metabolism of cells. The above-mentioned choline analogs containing azide groups are used to modify organisms. This method has little impact on the activity of organisms. Among them, choline belongs to the components of cell membranes and participates in the biological metabolism of organisms. Compared with the large-volume DBCO group, the azide group has a smaller volume and has less impact on the activity of organisms.

[0031] In step S01, the amou...

Embodiment 1

[0061] S11. Raw cell264.7 cells were modified with choline analogues: first, the Raw cell264.7 cells were washed twice with PBS buffer, and the above-mentioned corresponding fresh medium was replaced, and then choline with a final concentration of 1 μg / mL Alkaline analog modification reagent AE-Cho was co-cultured with Raw cell264.7 cells, specifically incubated in a 5% CO2 incubator at 37°C for 24 hours, then washed with PBS buffer to remove excess choline analog modification reagent, and added fresh culture medium, spare. Wherein, the molar ratio of Raw cell264.7 cells to choline analogues is 1:10.

[0062] S12. Labeling Raw cell264.7 cells with fluorescent dyes modified by DBCO groups: washing the cells modified with choline analogues obtained in the above step S11 with PBS buffer twice to remove residual choline analogue modification reagents; Then add DBCO-Fluor488, a DBCO-modified fluorescent dye whose molar mass is 10 times that of Raw cell264.7 cells, into the above c...

Embodiment 2

[0064] S21. Modification of dendritic cells with choline analogues: first wash the dendritic cells twice with PBS buffer, replace the corresponding fresh medium above, and then add choline with a final concentration of 0.5 μg / mL The analog modification reagent AE-Cho was co-cultured with dendritic cells, specifically incubate in a 5% CO2 incubator at 37°C for 24 hours, then wash off excess choline analog modification reagent with PBS buffer, and add fresh culture Base, spare. Wherein, the molar ratio of dendritic cells to choline analogs is 1:10.

[0065] S22. Label dendritic cells with fluorescent dyes modified by DBCO groups: wash the cells modified with choline analogues obtained in the above step S21 twice with PBS buffer to remove residual choline analogue modification reagents; then Add DBCO-Fluor488, a fluorescent dye modified by DBCO groups whose molar mass is 10 times that of dendritic cells, to the above cell culture medium, and incubate at 37°C for 1 hour to carry ...

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Abstract

The invention discloses a fluorescent labeling method for living organisms having cell membrane structures. The fluorescent labeling method comprises the following steps: co-culturing choline analogue containing azide group and a living organism having a cell membrane structure and acquiring choline analogue modified living organism after metabolism; and marking the choline analogue modified living organism by adopting DBCO (dibenzocyclooctyne) group modified fluorescent marker through the click chemical reaction and enabling the fluorescent marker to be labeled on the surface of the cell membrane of the living organism. According to the fluorescent labeling method for the living organism having the cell membrane structure, the choline analogue containing azide group is adopted to modify the living organism, and the method has little influence on the activity of the living organism; and the fluorescent labeling method adopts a one-step method for marking, compared with the two-step method, the one-step method can be operated simply and has high efficiency.

Description

technical field [0001] The invention belongs to the technical field of biomarkers, in particular to a fluorescent labeling method for organisms with cell membrane structures. Background technique [0002] At present, cell therapy (including stem cells and immune cells) is widely used in many clinical treatment fields such as tumors, autoimmune diseases, and transplantation. Studying the migration and distribution of cells in vivo is crucial for the clinical application and promotion of cell therapy. The existing cell labeling methods mainly use fluorescent dyes, antibodies (immunological fluorescent labels), reporter genes and fluorescein and other related labeling techniques to study cell morphology, cell fusion, cell dynamics, cell migration and other cell functions, and some The technology needs to be combined with histopathology-related slicing techniques. Tissues are taken out of animals at different time points for sectioning, staining and marking, and various physiol...

Claims

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Application Information

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IPC IPC(8): G01N33/58
CPCG01N33/582
Inventor 马轶凡潘宏张鹏飞李文军蔡林涛
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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