Method for detecting phosphorylation level of intracellular tyrosine

A technology of phosphorylation level and detection method, which is applied in measurement device, fluorescence/phosphorescence, preparation of test samples, etc., to achieve the effect of speeding up progress, simple operation steps, and saving time and cost

Pending Publication Date: 2020-08-11
SICHUAN BRANCH OF CHINA TOBACCO +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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At present, there is

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  • Method for detecting phosphorylation level of intracellular tyrosine
  • Method for detecting phosphorylation level of intracellular tyrosine
  • Method for detecting phosphorylation level of intracellular tyrosine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Flow cytometry was used to detect the level of intracellular tyrosine phosphorylation under exposure to low concentrations of smoke condensate in different smoking modes.

[0032] Smoke condensate preparation: extract cigarette samples according to GB / T 5606.1-2004, and use RM20H rotary table smoking machine to smoke under two smoking conditions of ISO and HCI respectively, the total particulate matter (Total Particulate Matter, TPM) use mm Cambridge filter discs were collected, and a corresponding volume of DMSO was added according to the quality of TPM to obtain a TPM stock solution with a final concentration of 10 mg / mL;

[0033] Cell infection: the cell density is 2.5×10 5 cells / mL were plated and cultured overnight. Add 120 μg / mL (respectively prepared under ISO and HCI suction conditions) of the cell culture medium of the smoke condensate for cell culture for 1 hour;

[0034] Immunofluorescence labeling and sample processing: the infected cells were washed th...

Embodiment 2

[0037] Flow cytometry was used to detect the level of intracellular tyrosine phosphorylation under exposure to high concentrations of smoke condensate in different smoking modes.

[0038] Smoke condensate preparation: extract cigarette samples according to GB / T 5606.1-2004, and use RM20H rotary table smoking machine to smoke under two smoking conditions of ISO and HCI respectively, the total particulate matter (Total Particulate Matter, TPM) use mm Cambridge filter discs were collected, and a corresponding volume of DMSO was added according to the quality of TPM to obtain a TPM stock solution with a final concentration of 10 mg / mL;

[0039] Cell infection: the cell density is 2.5×10 5 cells / mL were plated and cultured overnight. Add 300 μg / mL (respectively prepared under ISO and HCI suction conditions) of the cell culture medium of the smoke condensate for cell culture for 1 hour;

[0040] Immunofluorescence labeling and sample processing: the infected cells were washed thr...

Embodiment 3

[0045] The levels of intracellular tyrosine phosphorylation in cells exposed to low concentrations of smoke condensate in different smoking modes were detected by confocal laser microscopy.

[0046] Smoke condensate preparation: extract cigarette samples according to GB / T 5606.1-2004, and use RM20H rotary table smoking machine to smoke under ISO and HCI conditions respectively, TPM uses According to the mass of TPM, a corresponding volume of DMSO was added to obtain a TPM stock solution with a final concentration of 10 mg / mL.

[0047] Cell culture and treatment: Human oral epidermoid carcinoma KB cells were cultured in a-MEM complete medium containing 10% fetal bovine serum at 37°C, 5% CO 2 cultivated under conditions. When the cell confluency is 70%-80%, digest with trypsin, resuspend the medium to make a single cell suspension, and adjust the cell concentration to 4×10 4 cells / mL, inoculate 100 μL per well into a 96-well plate, and culture overnight. Add 40μg / mL flue gas...

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Abstract

The invention discloses a method for detecting the phosphorylation level of intracellular tyrosine. The method comprises the following steps: preparing a smoke condensate, treating cells, carrying outimmunofluorescence labeling and sample treatment, and detecting. By researching the concentration level of active oxygen in cells after smoke exposure, the influence of smoke exposure on oxidative stress reaction of cells is evaluated, biological evaluation is performed on the safety of smoke components, and scientific reference is reversely provided for research, development and production of safe cigarettes.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis of intracellular phosphorylated tyrosine, and relates to a detection method for intracellular phosphorylated tyrosine. Background technique [0002] Smoking causes multiple physiological reactions such as gene damage, abnormal protein expression, and abnormal metabolism of exogenous compounds. The apparent reflection is the occurrence of multiple biological effects, such as oxidative stress, inflammatory response, and intracellular tyrosine phosphorylation. [0003] Flow cytometry (FCM) is a cell analysis technology that integrates multiple disciplines and technologies such as optics, electronics, fluid mechanics, cytochemistry, immunology and computer science. It is not subject to molecular biology. Abnormal effects can be visualized for accurate quantification of intracellular phosphorylated tyrosine. [0004] Smoking causes multiple physiological reactions such as gene damage, abn...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N15/14G01N1/34
CPCG01N21/6428G01N21/6458G01N15/1434G01N1/34G01N2021/6439G01N2015/144
Inventor 范子彦熊巍杨飞陶晓秋王颖唐纲岭卫民庞夙
Owner SICHUAN BRANCH OF CHINA TOBACCO
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