Open AccessImmunofluorescent evaluation of DNA repair synthesis using interactive laser cytometry
Author(s) -
Srivastava Vinod,
Miller Susan,
Busbee David
Publication year - 1993
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990140206
Subject(s) - cytometry , flow cytometry , dna , microbiology and biotechnology , chemistry , biology , biochemistry
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 fluorochromelabeled 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 3 H‐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. © 1993 Wiley‐Liss, Inc.