title: Application of click chemistry conditions for 5-bromo-2'-deoxyuridine determination through Fenton and related reactions.
abstract: Mixtures of ascorbate and copper used in certain click chemistry experimental conditions act as oxidizing agents, catalyzing the formation of reactive oxygen species through Fenton and related reactions. Hydroxyl radicals act as chemical nucleases, introducing DNA strand breaks that can be exploited for BrdU immunostaining in place of acid denaturation. This procedure is readily applicable to high content analysis and flow cytometry assays, and provides results comparable to click chemistry EdU cycloaddition and classical BrdU immunodetection. Importantly, this approach allows preservation of labile epitopes such as phosphoproteins. This unit describes an optimized method that successfully employs Fenton chemistry for simultaneous detection of phosphoproteins and BrdU in intact cells.
authors.affiliation: Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Cappella
authors.affiliation: Department of Chemistry, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Pulici
authors.affiliation: Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Gasparri
mesh_terms: D001973:Bromodeoxyuridine / Q000378:metabolism*; D002384:Catalysis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D057930:Click Chemistry / Q000379:methods*; D004247:DNA / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007150:Immunohistochemistry; D007501:Iron / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: C045076:Fenton's reagent; D004247:DNA; D006861:Hydrogen Peroxide; D007501:Iron; D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine
keywords: BrdU; Fenton chemistry; click chemistry; high content analysis; sodium ascorbate