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Gamma H2AX based cell DNA damage detecting method

A DNA damage and H2AX technology, applied in measurement devices, biological tests, material inspection products, etc., can solve the problems of large loss of cells after repeated washing, non-specific fluorescence interference, complicated operation steps, etc., and achieve fast and convenient detection process. The operation method is simple and the effect of improving the detection efficiency

Inactive Publication Date: 2015-10-07
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these techniques have problems such as cumbersome operation steps, susceptibility to non-specific fluorescence interference, large loss of cells after repeated washing, and long test cycle.

Method used

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  • Gamma H2AX based cell DNA damage detecting method
  • Gamma H2AX based cell DNA damage detecting method
  • Gamma H2AX based cell DNA damage detecting method

Examples

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Effect test

example 1

[0027] Example 1 Evaluation of NNK

[0028] Collect CHO cells in the exponential growth phase at 1×10 6 Each cell / well was seeded in a 6-well plate, cultured in a cell incubator for 24 hours, and then exposed to NNK for 24 hours. In the experiment, the doses of NNK were 0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 400 μg / mL to infect CHO cells, and three parallel wells were set up for each condition , placed at 37 °C, 5% CO 2 cultured in a cell culture incubator for 24 h. After the cells were infected, add 500 μL of 0.25% trypsin, digest for 1-2 min, discard the trypsin, add 1 mL of phosphate buffered saline (PBS) to suspend, centrifuge at 1500 rpm / min, discard the supernatant, Wash twice. Ethanol fixation: Prepare 70% ethanol and pre-cool at -20°C. After vortexing to loosen the cell aggregates, 1 mL of pre-cooled 70% ethanol was added drop by drop while vortexing, and placed in a -20 °C refrigerator for 24 h. TritonX-100 membrane rupture: Take out the fixed sam...

example 2

[0031] Example 2 Evaluation of AαC

[0032] Collect CHO cells in the exponential growth phase at 1×10 6 Each cell / well was seeded in a 6-well plate, cultured in a cell incubator for 24 h, and then AαC was exposed for 24 h. In the experiment, the doses of AαC were 0 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL and 40 μg / mL to infect CHO cells, and three parallel wells were set up for each condition , placed at 37 °C, 5% CO 2 cultured in a cell culture incubator for 24 h. After the cells were infected, add 500 μL of 0.25% trypsin, digest for 1-2 min, discard the trypsin, add 1 mL of phosphate buffered saline (PBS) to suspend, centrifuge at 1500 rpm / min, discard the supernatant, Wash twice. Ethanol fixation: Prepare 70% ethanol and pre-cool at -20°C. After vortexing to loosen the cell aggregates, add 1 mL of pre-cooled 70% ethanol drop by drop while vortexing, and place in a -20 °C refrigerator overnight for fixation. TritonX-100 membrane rupture: Take out the fixed sample...

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Abstract

The invention discloses a gamma H2AX based cell DNA damage detecting method. The gamma H2AX based cell DNA damage detecting method is characterized by comprising the following technological steps: 1) preparing an experiment reagent; 2) culturing cells; 3) contaminating the cells; 4) determining gamma H2AX in the cells by a flow cytometer; and 5) acquiring a result and analyzing the result. Compared with the prior art, the gamma H2AX based cell DNA damage detecting method has the following characteristics: in the whole testing process, the testing steps of washing cells for multiple times are reduced, so that the operation method is relatively simple and convenient; aiming at the problem of relatively high nonspecific fluorescence interference in an indirect marking method, a direct immune fluorescence marking method is established, so that the detection process is relatively rapid and convenient, and the detection result is relatively stable; according to the invention, the steps of marking a first antibody and a second antibody in an indirect marking method are omitted, the detection cycle is shortened, and the detection efficiency is enhanced. The flow cytometer is used for detecting the relative fluorescence intensity of gamma H2AX, the result is relatively objective and accurate, and the statistic precision is high.

Description

technical field [0001] The invention relates to a method for detecting cell DNA damage marker γH2AX, which is used for genotoxicity detection of compounds in cigarette smoke, specifically a method for detecting cell DNA damage based on γH2AX. Background technique [0002] Severe cellular DNA damage mainly occurs in the following situations: 1) double-strand breaks (DSBs) induced directly by the cell under the action of physical, chemical or biological factors or induced by replication stress, 2) the process of cell self-regulation DSBs, 3) DSBs produced during retroviral transfection of cells. The DNA damage in the above three situations can induce the phosphorylation of γH2AX (CH2AX) and the clustering to form focal points. This process can occur within a few minutes. The number of focal points formed by γH2AX is related to the number of DNA double-strand breaks There is a one-to-one correspondence. Therefore, γH2AX is considered to be a specific indicator for detecting D...

Claims

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Application Information

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IPC IPC(8): G01N15/14G01N33/58
Inventor 赵俊伟李翔谢复炜康彧赵阁尚平平刘惠民谢剑平
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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