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A kind of ATP detection reagent of 3D organoid and its preparation method and application

A detection reagent, NP-40 technology, applied in the field of biomedicine, can solve the problems of poor linearity, poor accuracy, poor repeatability, etc., and achieve the effect of reducing fluorescence quenching, enhancing duration, and improving repeatability and accuracy

Active Publication Date: 2021-06-11
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Organoids are a kind of in vitro 3D culture cultured by stem cells simulating the in vivo microenvironment. With the development of 3D cultured organoid technology, ordinary ATP chemiluminescent detection reagents show poor reproducibility when applied to the detection of cell viability in 3D organoids. , poor linearity, and poor accuracy have been unable to meet the needs of 3D organoid cell viability detection

Method used

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  • A kind of ATP detection reagent of 3D organoid and its preparation method and application
  • A kind of ATP detection reagent of 3D organoid and its preparation method and application
  • A kind of ATP detection reagent of 3D organoid and its preparation method and application

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Embodiment 1

[0024] This embodiment provides an ATP detection reagent, wherein the final concentration of each component is composed of: firefly luciferin 45mg / ml, North American firefly luciferase 80ug / ml, p-iodophenol 2.0mM, quinol dimethacrylate 4.5 mM, PBS 0.1M, Tris 50mM, NP-40 0.2%, sodium deoxycholate 2.0%, Tween 0.2%, CHAPS 2.0%, guanidine hydrochloride 0.1M, DDT 15mM, wherein percentages are weight percentages. Its preparation method is as follows: dosing according to the final concentration of the above components; first dissolve PBS and Tris in sterile water to make a mixed buffer solution; mix firefly luciferin, North American firefly luciferase, p-iodophenol, quinol di Methacrylate, NP-40, sodium deoxycholate, Tween-40, CHAPS, guanidine hydrochloride, and DDT were dissolved in the mixed buffer solution to obtain the obtained product.

Embodiment 2

[0026] This embodiment provides an ATP detection reagent, wherein the final concentration of each component is composed of: firefly luciferin 65mg / ml, North American firefly luciferase 60ug / ml, p-iodophenol 4.5mM, quinol dimethacrylate 2.0 mM, PBS 0.2M, Tris 30mM, NP-40 2.0%, sodium deoxycholate 0.2%, Tween 2.0%, CHAPS 0.2%, guanidine hydrochloride 0.3M, DDT 10mM, wherein percentages are weight percentages. The preparation method is the same as in Example 1.

Embodiment 3

[0028] This embodiment provides an ATP detection reagent, wherein the final concentration of each component is composed of: firefly luciferin 50mg / ml, North American firefly luciferase 70ug / ml, p-iodophenol 3.5mM, quinol dimethacrylate 3.5 mM, PBS 0.15M, Tris 40mM, NP-40 1.2%, sodium deoxycholate 1.2%, Tween 1.2%, CHAPS 1.2%, guanidine hydrochloride 0.2M, DDT 12mM, wherein percentages are weight percentages. The preparation method is the same as in Example 1.

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Abstract

The invention provides an ATP detection reagent and its preparation method and application. The reagent includes firefly luciferin, North American firefly luciferase, p-iodophenol, quinol dimethacrylate, PBS, Tris, NP‑40, deoxygenated Sodium cholate, Tween, CHAPS, guanidine hydrochloride, DDT and sterile water. The preparation method includes: dissolving PBS and Tris in sterile water first to make a mixed buffer; mixing firefly luciferin, North American firefly luciferase, p-iodophenol, quinol dimethacrylate, NP‑40, deoxygenated Sodium cholate, Tween‑40, CHAPS, guanidine hydrochloride, and DDT were dissolved in the mixed buffer. The detection reagent of the invention can fully meet the requirements of 2D and 3D cell line ATP detection, and also has good repeatability and accuracy in the aspect of 3D organoid cell ATP detection. In addition, the detection reagent of the present invention can enhance the duration of chemiluminescence and reduce fluorescence quenching.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an ATP detection reagent and its preparation method and application. Background technique [0002] ATP chemiluminescence detection method is a cell viability detection method that has emerged in recent years. When cells are damaged, the synthesis of ATP in the cells decreases, and when cells die, ATP will quickly hydrolyze and disappear. Therefore, the cell viability and the number of living cells can be detected by measuring the endogenous ATP content of the cells. [0003] When the chemiluminescence method is used to detect the endogenous ATP of the cells, the cells to be tested need to be ruptured first, and the ATP in the cells is released to fully react with the luminescent reagent. The stability of ATP molecules is poor, and it is easy to be inactivated. Therefore, the key factors for accurate detection of cell viability are to rupture cells as soon as poss...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/66C12Q1/00
CPCC12Q1/008C12Q1/66C12Q2326/50
Inventor 尹天武李刚陈泽新
Owner ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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