Immunofluorescent evaluation of DNA repair synthesis using interactive laser cytometry.
An improved differential fluorescence analysis technique which employs scanning laser cytometry for the detection of two DNA binding fluorochromes was used to quantitate both total DNA and cellular incorporation of the base analogue, bromodeoxyuridine (BrdUrd), into UV- or methyl methane sulphonate (MMS)-treated DNA. In this procedure DNA containing BrdUrd was partially denatured and immunolabeled by binding anti-BrdUrd monoclonal IgG to incorporated BrdUrd. Anti-BrdUrd was detected with a secondary fluorochrome-labeled polyclonal anti-IgG. Cells were counterstained with propidium iodide to allow the determination of total DNA. Fluorescence was determined using the Meridian ACAS 570 Scanning Laser Cytometer. The computer-analyzed images were generated from monodisperse populations of UV- or MMS-treated cells, allowing the evaluation of DNA synthesis associated with excision repair and of total DNA content in single cells. These data were compared with 3H-thymidine incorporation occurring as a function of excision repair. The analysis of DNA synthesis with this technique is consistent, relatively simple, and rapid with excellent sensitivity and provides a viable method for determining cycling vs. non-cycling cells, total cellular DNA, and excision repair-associated DNA synthesis of individual cells within heterogeneous cell populations.
Srivastava
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V
Department of Anatomy and Public Health, College of Veterinary Medicine, Texas A & M University, College Station 77843.
Miller
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S
Busbee
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D
D001973:Bromodeoxyuridine; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000096:biosynthesis*; D004260:DNA Repair; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007834:Lasers; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D004247:DNA; D001973:Bromodeoxyuridine