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Fluorescent probe for covalently connecting labeled cells and method for tracking labeled cells

A technology of fluorescent probes and linking labels, which is applied in the field of cell fluorescent probes, can solve the problems of small molecule fluorescent probe loss and short fluorescence retention time, and achieve the effects of easy promotion, cheap raw materials, and simple synthesis process

Inactive Publication Date: 2019-04-19
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Small molecule fluorescent probes are prone to bleed from biological samples, resulting in short fluorescence retention times

Method used

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  • Fluorescent probe for covalently connecting labeled cells and method for tracking labeled cells
  • Fluorescent probe for covalently connecting labeled cells and method for tracking labeled cells
  • Fluorescent probe for covalently connecting labeled cells and method for tracking labeled cells

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Preparation of red, green and blue covalently labeled fluorescent probes of the present invention

[0038] 1) Take a 50ml round bottom flask, add 3.4g, 20mmol 2-hydroxy-2-(4-hydroxyphenyl)acetic acid (mandelic acid, figure 2 Compound I), dissolved in 10ml of pyridine. Add 100 mmol acetyl chloride dropwise and stir at room temperature for 1 h. Then the solvent was spin-dried and purified by silica gel column to obtain figure 2 Compound II, yield 86%.

[0039] 2) Dissolve 504mg, 2.0mmol of compound II in 5ml of dichloromethane. 500 mg, 2.6 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 606 mg, 6.0 mmol triethylamine were added. Then add 914mg, 2.0mmol compound rhodamine derivatives (note figure 2 , Compound III). After 2 hours of reaction, it was treated with 1M dilute hydrochloric acid and extracted with dichloromethane. The organic phase was spin-dried and the solvent was purified through the column to obtain a red covalently l...

Embodiment 2

[0041] Example 2: A cell experiment in which the red covalently labeled fluorescent probe of the present invention is retained in cells for a long time.

[0042] 1) Weigh 2.5 mg, about 4 μmol of red covalently labeled fluorescent probe and dissolve in 1 mL of dimethyl sulfoxide. A standard solution of 4 mM red covalently labeled fluorescent probe was obtained.

[0043] 2) Mix 10 μL of standard solution of red covalently labeled fluorescent probe with 10 mL of cell culture solution to obtain cell culture medium containing 4 μmol / L (4 μM) of red covalently labeled fluorescent probe.

[0044] 3) Incubate the cell culture medium containing 4 μM red covalently labeled fluorescent probe with HeLa cells in a 35 mm glass bottom culture dish for 1 hour, wash with fresh cell culture medium three times, and use confocal A fluorescence microscope collects intracellular fluorescent signals. See the experimental results image 3 , indicating that the signal of the probe still remained af...

Embodiment 3

[0046] Example 3: The combination of red, green and blue covalently labeled fluorescent probes of the present invention is applied to the experiment of tracking and marking cells in a multi-cell system

[0047] 1) Same as Steps 1 and 2 of Example 2, to obtain standard cell culture solution of red, green and blue covalently labeled fluorescent probes at a concentration of 4 μM.

[0048]2) Incubate the HeLa cells with the cell culture medium containing 4 μM red, green, and blue covalently labeled fluorescent probes respectively in wells of a 24-well plate, and incubate for 1 h. Wash 3 times with fresh cell culture medium. The three kinds of untreated cells were mixed and subcultured and seeded in the same 35mm glass-bottom culture dish. After 24 hours, intracellular fluorescence signals were collected with a confocal fluorescence microscope. See the experimental results Figure 5 , indicating that the covalently labeled fluorescent probe of the present invention can be applie...

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Abstract

The invention discloses a fluorescence probe for covalently connecting labeled cells and a method for tracking labeled cells, and relates to a cell fluorescence probe, wherein the fluorescent probe for covalently connecting labeled cells consists of a covalent connecting part and a fluorescent group part; the fluorescent group can be any fluorescent group which can be a derivative of any of the fluorescent groups reported in the literature which can be selected from one of rhodamine, coumarin, fluorescein and the like, bonding to the covalently linked moiety is achieved by drawing a primary orsecondary amine group on the fluorescent group, thereby achieving multicolor, multifunctional covalently labeled cells. The specificity of covalently labeled cells is that the fluorescent probe is only capable of reacting with intracellular proteins and not with proteins in the cell culture medium. The covalent linking moiety is covalently labeled as a probe that forms a chemical bond with the intracellular protein. The fluorescence probe can be used for tracking and labeling cells specifically for a long time, and has low cytotoxicity.

Description

technical field [0001] The invention relates to a fluorescent probe for cells, in particular to a fluorescent probe covalently linked to marked cells and a method for tracking the marked cells. Background technique [0002] Fluorescence imaging technology has become a real-time, high-resolution, non-invasive method to monitor biological processes, in which chemical small molecule probes play a very important role. Small molecule fluorescent probes are prone to bleed from biological samples, resulting in short fluorescence retention times. Fluorescent probes linked to biomacromolecules in biological samples through covalent bonds have a longer retention time in biological samples and more stable fluorescent signals. [0003] Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a covalently linked fluorescent probe widely used in the study of biological processes. Commercial Cell-Trackers including CFSE not only bind to intracellular Proteins are reactive and readily re...

Claims

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Application Information

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IPC IPC(8): C07D311/82C09K11/06G01N15/14G01N15/10G01N21/64
CPCC07D311/82C09K11/06C09K2211/1007C09K2211/1044C09K2211/1088G01N15/10G01N15/14G01N21/6428G01N21/6486G01N2021/6439
Inventor 韩守法施一龙朱瑞
Owner XIAMEN UNIV
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