Application of Click Chemistry Conditions for 5‐Bromo‐2′‐Deoxyuridine Determination Through Fenton and Related Reactions. Issue 1 (5th January 2015)

• Record Type: Journal Article
• Authors: Cappella, Paolo; Pulici, Maurizio; Gasparri, Fabio
• Editors: Robinson, J. Paul; Darzynkiewicz, Zbigniew; Hoffman, Robert; Nolan, John P.; Rabinovitch, Peter S.; Watkins, Simon
• Is Part Of: Current protocols in cytometry. Volume 71:Issue 1(2015)
• Page Start: 7.43.1
• Page End: 7.43.17
• Publication Date: 2015-01-05
• Subjects: sodium ascorbate -- BrdU -- click chemistry -- Fenton chemistry -- high content analysis; Cytology -- Laboratory manuals; Flow cytometry -- Laboratory manuals; Cell separation -- Laboratory manuals; Molecular biology -- Laboratory manuals; Flow Cytometry -- methods; Image Cytometry -- methods; Cell Separation -- methods; Cytological Techniques; Molecular Biology -- methods; Cell separation; Cytology; Flow cytometry; Molecular biology; Laboratory Manuals; Laboratory manuals; 571.6
• DOI: 10.1002/0471142956.cy0743s71
• Languages: English
• ISSNs: 1934-9297

Abstract

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. © 2015 by John Wiley & Sons, Inc.