To examine the expression level of γH2AX in the nucleus, cells attached to the coverslip were treated with H2O2 in the presence or absence of mangiferin, fixed with 2% paraformaldehyde, and then permeabilized with 0.1% Triton-X-100 as previously described (Park et al., 2023 (link)). The cells were probed with anti-γH2AX antibody (Ser139, Thermo Fisher Scientific) and then reacted with Alexa Fluor 594-conjugated antibody (Cell Signaling Technology). After counterstaining the nuclei using 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), fluorescence images were acquired under a fluorescence microscope.
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Quantifying γH2AX Levels in Cells
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Corresponding Organization :
Other organizations : Dong-Eui University, Kosin University, Pusan National University, Chungbuk National University, Chung-Ang University, Jeju National University, Mokpo National University, China-US (Henan) Hormel Cancer Institute
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Variable analysis
independent variables
- H2O2 treatment
- Mangiferin treatment
- Expression level of γH2AX in the nucleus
- Cells attached to the coverslip
- Fixation with 2% paraformaldehyde
- Permeabilization with 0.1% Triton-X-100
- Probing with anti-γH2AX antibody (Ser139, Thermo Fisher Scientific)
- Reaction with Alexa Fluor 594-conjugated antibody (Cell Signaling Technology)
- Counterstaining of nuclei with DAPI (Sigma-Aldrich)
- Fluorescence microscopy imaging
- Positive control: Not specified
- Negative control: Not specified
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