title: DNA Fiber Spreading Assay to Test HDACi Effects on DNA and Its Replication.
abstract: DNA fiber spreading assay is an invaluable technique to visualize and follow the spatial and temporal progress of individual DNA replication forks. It provides information on the DNA replication progress and its regulation under normal conditions as well as on replication stress induced by environmental genotoxic agents or cancer drugs. The method relies on the detection of incorporated thymidine analogues during DNA synthesis in the S phase of the cell cycle by indirect immunofluorescence. Here, we describe the procedure established in our laboratories for sequential pulse labeling of human cells with 5-chloro-2'-deoxyuridine (CldU) and 5-iodo-2'-deoxyuridine (IdU), cell lysis, and DNA fiber spreading on slides and sequential immunodetection of the incorporated thymidine analogues by primary antibodies recognizing specifically CldU or IdU alone. We describe also the laser scanning imaging, classification, and measurement of the detected DNA fiber tracks. The obtained quantitative data can be evaluated statistically to reveal the immediate or long-term effects of DNA-damaging agents, DNA repair inhibitors, and epigenetic modulators like HDAC inhibitors on DNA replication in normal and tumor cells.
authors.affiliation: Institute of Toxicology, University Medical Center Mainz, Mainz, Germany. nikolova@uni-mainz.de.
authors.forename: Teodora
authors.initials: T
authors.lastname: Nikolova
authors.affiliation: Institute of Toxicology, University Medical Center Mainz, Mainz, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Göder
authors.affiliation: Laboratory of Radiobiology Experimental Radiooncology, Clinic of Radiotherapy and Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Ann
authors.initials: A
authors.lastname: Parplys
authors.affiliation: Laboratory of Radiobiology Experimental Radiooncology, Clinic of Radiotherapy and Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Borgmann
mesh_terms: D000906:Antibodies / Q000737:chemistry; D001549:Benzamides / Q000494:pharmacology; D001681:Biological Assay; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004261:DNA Replication; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods; D045325:HCT116 Cells; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007065:Idoxuridine / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D011725:Pyridines / Q000494:pharmacology; D016196:S Phase; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000906:Antibodies; D001549:Benzamides; D056572:Histone Deacetylase Inhibitors; D011725:Pyridines; C118739:entinostat; C012244:5-chloro-2'-deoxyuridine; D004247:DNA; D006655:Histone Deacetylases; D007065:Idoxuridine; D003857:Deoxyuridine
keywords: Anti-BrdU antibody; DNA fiber spreading; Immunodetection; LSM microscopy; Thymidine analogue labeling