An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). Although several methods are available for the bio-chemical analysis of this molecule, its determination at the single cell level may provide significantadvantages when investigating the influence of cell heterogeneity and cell type in the DNA damageresponse. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detectionwith the glycoprotein avidin has also been proposed because of a structural similarity between itsnatural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliabilityand sensitivity is not clear. In this study, we compared the immunofluorescence determination of8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugatedwith the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different celltypes by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species(ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, ourresults indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greaterthan that attained with avidin-AF488. These findings suggest that immunofluorescence techniquesare best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage.
Single Cell Determination of7,8-dihydro-8-oxo-2′-deoxyguanosine by FluorescenceTechniques: Antibody vs. Avidin Labeling
Giulio Ticli;Ornella Cazzalini;Lucia A. Stivala;
2023-01-01
Abstract
An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). Although several methods are available for the bio-chemical analysis of this molecule, its determination at the single cell level may provide significantadvantages when investigating the influence of cell heterogeneity and cell type in the DNA damageresponse. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detectionwith the glycoprotein avidin has also been proposed because of a structural similarity between itsnatural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliabilityand sensitivity is not clear. In this study, we compared the immunofluorescence determination of8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugatedwith the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different celltypes by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species(ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, ourresults indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greaterthan that attained with avidin-AF488. These findings suggest that immunofluorescence techniquesare best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
