Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

Summary

This protocol describes an efficient method for quantitatively detecting DNA oxidative damage in MCF-7 cells by ELISA technology.

Abstract

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly impacts gene expression and serves as a pivotal factor in stimulating neurodegenerative disorders, cancer, and aging. Therefore, precise quantification of 8-oxoG has clinical significance in the investigation of DNA damage detection methodologies. However, at present, the existing approaches for 8-oxoG detection pose challenges in terms of convenience, expediency, affordability, and heightened sensitivity. We employed the sandwich enzyme-linked immunosorbent assay (ELISA) technique, a highly efficient and swift colorimetric method, to detect variations in 8-oxo-dG content in MCF-7 cell samples stimulated with different concentrations of hydrogen peroxide (H₂O₂). We determined the concentration of H₂O₂ that induced oxidative damage in MCF-7 cells by detecting its IC₅₀ value in MCF-7 cells. Subsequently, we treated MCF-7 cells with 0, 0.25, and 0.75 mM H₂O₂ for 12 h and extracted 8-oxo-dG from the cells. Finally, the samples were subjected to ELISA. Following a series of steps, including plate spreading, washing, incubation, color development, termination of the reaction, and data collection, we successfully detected changes in the 8-oxo-dG content in MCF-7 cells induced by H₂O₂. Through such endeavors, we aim to establish a method to evaluate the degree of DNA oxidative damage within cell samples and, in doing so, advance the development of more expedient and convenient approaches for DNA damage detection. This endeavor is poised to make a meaningful contribution to the exploration of associative analyses between DNA oxidative damage and various domains, including clinical research on diseases and the detection of toxic substances.

Introduction

DNA oxidative damage is a consequence of an imbalance between the generation of reactive oxygen species (ROS) and the cellular antioxidant defense system¹. It primarily involves the oxidation of DNA purine and pyrimidine bases^2,3. This oxidative modification of DNA bases not only compromises the integrity of the genome but also encompasses a wide range of pathological issues, including cancer, neurodegenerative diseases, and cardiovascular diseases^4,5. The guanine base in DNA has the lowest reduction potential and is the most su....

Protocol

1. Construction of an H₂O₂ -induced DNA oxidative damage model in MCF-7 cells

1. MCF-7 cell recovery
   1. Transfer the cell culture cryogenic tube, which contains 3.5 x 10⁶ MCF-7 cells and is stored in a -80°C refrigerator, rapidly to a foam box containing liquid nitrogen. Retrieve the tube with forceps and place it in a 37 °Cconstant temperature water bath for approximately 1 min to thaw the preserved cells.
      NOTE: During.......

Discussion

The development of ELISA methods holds great importance for both existing and new DNA damage detection methodologies. In comparison to traditional HPLC and mass spectrometry techniques, this approach not only is user-friendly but also exhibits high sensitivity and meets the demands of high-throughput screening³⁰. This enables the monitoring of 8-oxo-dG in large-scale disease screening studies, facilitating a deeper understanding of the correlation between this biomarker and various diseases.

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Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA(1x)	Gibco	25200-072
Cell Counting Kit-8	Dojindo	CK04
Cell Counting Plate	QiuJing	XB-K-25
CO2 incubator	Thermo	51032872
DMEM basic(1X)	Gibco	C11995500BT
FBS	PAN	ST30-3302
GraphPad Prism X9	GraphPad Software		statistical analysis software
H2O2(3%)	Jiangxi Caoshanhu Disinfection Co.,Ltd.	1028348
high-speed centrifuge	Thermo	9AQ2861
Human 8-oxo-dG ELISA Kit	Zcibio	ZC-55410
L-1000XLS+ Pipettes	Rainin	17014382
L-20XLS+ Pipettes	Rainin	17014392
liquid nitrogen tank	Mvecryoge	YDS-175-216
MCF-7 CELL	BNCC	BNCC100137
Multiskan FC microplate photometer	Thermo	1410101
PBS	Solarbio	P1020
Penicillin-Streptomycin Solution, 100X	Beyotime	C0222
Trinocular live cell microscope	Motic	1.1001E+12
Ultra-low temperature freezer	Haire	V118574