Rapid quantitative senescence cell detection method based on senescence-associated beta-galactosidase

A technology of galactosidase and senescent cells is applied in the field of rapid quantitative senescent cell detection based on senescence-related β-galactosidase, which can solve the problems of time-consuming counting, different color shades, harsh experimental conditions, etc. The effect of reducing operation steps and reducing contact

Active Publication Date: 2019-05-24
ANHUI GUJING DISTILLERY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many detection methods for cell senescence, but most of them require the use of more expensive reagents, and the steps are complicated and demanding on experimental conditions; relatively low-cost detection methods mostly use the color development of reaction products to count the number of senescent cells, count It is time-consuming, and subjectivity is strong, and the color depth is different. The commonly used counting method ignores the relationship between the color depth of the product and the degree of cell aging. Chemical treatment establishes a method for rapid quantitative detection of cellular senescence

Method used

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  • Rapid quantitative senescence cell detection method based on senescence-associated beta-galactosidase
  • Rapid quantitative senescence cell detection method based on senescence-associated beta-galactosidase
  • Rapid quantitative senescence cell detection method based on senescence-associated beta-galactosidase

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Experimental program
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Effect test

Embodiment 1

[0041] Take out from the incubator with 25cm 2 Human gastric epithelial cells (Ges-1) that have been cultured for 72 hours in the cell culture flask were discarded, and the cells were washed 3 times with PBS, 2 mL each time; 2 After incubating in the incubator for 48 hours, take out the cell culture bottle, blow and blow the bottom of the culture bottle, transfer all the liquid into a centrifuge tube, centrifuge at 11000g for 5 minutes to obtain a blue product, add 300μL DMSO to dissolve the blue product, and heat at 37°C for 1 hour. After it is completely dissolved, Centrifuge at 14000g for 3min, pipette 200μL supernatant into a 96-well plate, measure the absorbance value at 640nm, the absorbance value is 0.573, the absorbance is linearly correlated with the number of senescent cells, each 25cm 2 The number of senescent cells in the cell culture flask is about 470,000.

Embodiment 2

[0043] Take out Ges-1 that has been cultured for 48 hours in a 6-well plate from the incubator, discard the medium, add 1 mL of PBS to wash the cells twice, then add 2 mL of cell senescence staining solution, and store at 37 °C without CO 2 Incubate in an incubator for 72 hours, take out the cell culture bottle, blow the bottom of the culture bottle, transfer all the liquid to a centrifuge tube, centrifuge at 12,000g for 4 minutes to obtain a blue product, add 220 μL DMSO to it to dissolve the blue precipitate, heat at 38°C for 1 hour, and wait until it is completely dissolved Afterwards, centrifuge at 14000g for 4min, pipette 200μL supernatant into a 96-well plate, measure the absorbance value at 640nm, the absorbance value is 0.326, the absorbance is linearly correlated with the number of senescent cells, and the number of senescent cells is about 230000.

[0044] Table 1 The relationship between the number of senescent cells and their corresponding absorbance

[0045] ...

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Abstract

The invention discloses a rapid quantitative senescence cell detection method based on senescence-associated beta-galactosidase. Culture solution is discarded from in-vitro cultured adherent cells, the adherent cells are cleaned by PBS (phosphate buffer solution), staining solution is added, the adherent cells are placed into a 37 DEG C carbon-dioxide-free incubator, all the staining solution is transferred into a centrifugal tube after staining and then discarded after centrifugation, a blue product is cleaned by the PBS, DMSO (dimethyl sulfoxide) is added to dissolve the blue product, the blue product is centrifuged, supernatant is taken, the absorbance of the solution at a 640nm position is measured, a standard curve between the number of senescence cells and the absorbance is built bythe same method, and the number of the senescence cells of a test sample can be known according to the standard curve after the absorbance is measured. The method has the advantages of high reproducibility and reliability, strong objectivity, fewer influence factors, high speed and the like, and animal and human senescence cells can be rapidly quantified.

Description

technical field [0001] The invention relates to a rapid quantitative senescent cell detection method based on senescence-related β-galactosidase, which uses a microplate reader to determine the quantity of senescent cells through the absorbance of the color of a solution with a certain thickness. Background technique [0002] With the people's growing needs for a better life, more and more people begin to pay attention to health; the demand for anti-oxidation and anti-aging also increases. Correspondingly, with the continuous development of various technologies and the urgent needs of the people, research on anti-oxidation and anti-aging has gradually increased, including the effects of drug use, external stimuli, and special foods on cell aging. There are many detection methods for cell senescence, but most of them require the use of more expensive reagents, and the steps are complicated and demanding on experimental conditions; relatively low-cost detection methods mostly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/40C12Q1/06
Inventor 吴翠芳刘国英何宏魁李静心袁志强李安军
Owner ANHUI GUJING DISTILLERY
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