This research aims to quantitatively measure 8-oxo-dG in MCF-7 cells using ELISA, assessing the impacts of hydrogen peroxide on DNA oxidative damage. Specifically, ELISA steps are detailed in this video for efficiency. Techniques for detecting 8-Oxo-dG, including high-performance liquid chromatography with electrochemical detection, mass spectrometry, and other sophisticated methodologies have demonstrated efficacy.
However, these matters often involve numerous tedious steps and can be quite costly. This protocol addresses the research gap in efficient and sensitive detection of oxidative DNA damage, specifically 8-oxo-dG in cell samples, which is crucial for understanding its implications in disease, aging, and response to environmental stresses. My lab focused on method innovation for DNA damage event detection.
This will enable us to conduct in-depth life science research on DNA damage markers. After generating hydrogen peroxide-induced MCF-7 cell culture, discard the supernatant from the culture dish. Add one milliliter of PBS and gently shake the dish before discarding the PBS.
Now, add one milliliter of trypsin to wash the cells. Shake the dish and wait for one minute. Then discard trypsin.
Gently tap the dish. Then observe the cell movement in the sheet to confirm detachment. Next, add four milliliters of complete culture medium and transfer the cell suspension to a 15-milliliter centrifuge tube.
Centrifuge the suspension at 800G for five minutes and remove the supernatant. Add 200 microliters of PBS to resuspend the pellet and transfer the cell suspension to a 1.5 milliliter tube. After that, perform repeated freeze-thaw cycles to disrupt the cells.
Centrifuge the cell lysate at 1, 500G for 10 minutes and collect the supernatant for analysis. Thaw ELISA reagents at room temperature for 20 minutes before starting the assay. Preheat the ELISA reader 15 minutes before use.
Dilute the required 20-times concentrated wash buffer with deionized water into a working solution. Prepare working solutions of standard samples and carefully mix them up and down without bubble formation. Add 50 microliters of standard working solution and detection samples with different concentrations to the reaction wells excluding blank wells.
Add 100 microliters of horseradish peroxidase-conjugated detection antibody to each reaction well. Seal the wells with a plate seal and incubate at 37 degrees Celsius for 60 minutes. After incubation, remove the antibody solution.
Shake off the remaining liquid in the wells and pat dry on absorbent paper. Add 350 microliters of wash buffer to each reaction well. After one to two minutes, discard the buffer before adding 50 microliters of substrates A and B to each reaction well.
Seal the plate and incubate it at 37 degrees Celsius in the dark. After 15 minutes, add 50 microliters of stop solution to each reaction well. Immediately measure the optical density value at 450 nanometers wavelength with an ELISA reader.
The effect of hydrogen peroxide on the viability of MCF-7 cells demonstrated that treatment with 0.75 millimolar for 12 hours reduced the viability to 67%At a higher concentration of 1.5 millimolar, cell viability drastically dropped to below 3%MCF-7 cells following increasing concentration of hydrogen peroxide treatment showed increased levels of 8-Oxo-dG, illustrating the impact of oxidative stress on DNA damage.
