Use of the single-cell gel electrophoresis assay for the immunofluorescent detection of specific DNA damage.
The single-cell gel electrophoresis assay or comet assay is now a widely used method to assess the level of DNA damage in irradiated or chemically modified cells. We propose an adaptation of the currently applied protocol, aimed at singling out a defined modified base, using an immunodetection approach. After the electrophoresis step, the DNA tail moment was measured using ethidium bromide. Simultaneously, cyclobutane pyrimidine dimers (CPDs), the targeted lesions, were revealed by an indirect immunofluorescence detection using a specific monoclonal antibody. The assay was validated on human fibroblasts exposed to UVB light. The dose-response curves were established, showing a linear increase of the antibody response with the dose between 1000 and 10,000 J/m2. The detection limit of the method was 500 J/m2. Digestion of the CPDs, induced at 3000 J/m2, with T4 endonuclease V led to a marked decrease of the antibody response, confirming the specificity of the assay. A preliminary repair experiment is reported in which the tail moment of the comets together with the antibody response are measured, showing the disappearance of 80% of the antibody fixation sites within 48 h.
Sauvaigo
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Département de Recherche Fondamentale sur la Matière Condensée, SCIB/LAN, CEA Grenoble, France.
Serres
C
C
Signorini
N
N
Emonet
N
N
Richard
M J
MJ
Cadet
J
J
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D004247:DNA / Q000032:analysis* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D011740:Pyrimidine Dimers / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D011740:Pyrimidine Dimers; D004247:DNA