Application of Click Chemistry Conditions for 5‐Bromo‐2′‐Deoxyuridine Determination Through Fenton and Related Reactions

Paolo Cappella1, Maurizio Pulici2, Fabio Gasparri1

1 Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, 2 Department of Chemistry, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.43
DOI:  10.1002/0471142956.cy0743s71
Online Posting Date:  January, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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.

Keywords: sodium ascorbate; BrdU; click chemistry; Fenton chemistry; high content analysis

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Cytometry Analysis of Proliferating Cells and DNA Content using Fenton Chemistry
  • Basic Protocol 2: Flow Cytometry Analysis of Proliferating Cells and DNA Content Using BrdU/EdU Dual Labeling
  • Alternate Protocol 1: Fluorescence Immunocytochemistry and Image Acquisition using a High Content Analysis (HCA) Reader
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Flow Cytometry Analysis of Proliferating Cells and DNA Content using Fenton Chemistry

  Materials
  • 3.25 mM (1 mg/ml) 5‐bromo‐2′‐deoxyuridine (BrdU; Sigma, cat. no. B5002) in water (store up to 6 months at −20°C)
  • Cells cultured in appropriate culture medium
  • Phosphate‐buffered saline (PBS) without calcium and magnesium (Invitrogen)
  • 0.25% trypsin/0.02% EDTA solution (Invitrogen), 37°C
  • Serum‐containing medium
  • 100% ethanol or methanol (Sigma)
  • 0.1% Triton X‐100 (Sigma) in PBS
  • 1 M sodium ascorbate solution (see recipe)
  • Milli‐Q‐grade distilled water
  • 0.1 M Cu(II) sulfate solution (see recipe)
  • PBST (see recipe)
  • 0.025 mg/ml anti‐BrdU monoclonal mouse antibody (clone B44, BD Biosciences)
  • Alexa Fluor 488−conjugated goat anti‐mouse IgG (H+L; Invitrogen, cat. no. A‐11001)
  • Propidium iodide (PI) staining solution (see recipe)
  • Flow cytometer equipped with an Ar/He‐Ne dual laser for blue/red excitation (e.g., Beckton Dickinson BD FACSCalibur)
  • 37°C incubator
  • 15‐ml screw‐cap centrifuge tubes
  • Coulter counter
  • 12‐well tissue culture plate
  • Shaker
  • 5‐ml (12 × 75–mm) polystyrene tubes (BD Falcon)

Basic Protocol 2: Flow Cytometry Analysis of Proliferating Cells and DNA Content Using BrdU/EdU Dual Labeling

  Materials
  • 10 mM 5‐ethynyl‐2′‐deoxyuridine (EdU, Berry & Associates, cat. no. PY7562) in DMSO (store at –20°C) (optional)
  • Click‐IT EdU Alexa Fluor 488 Flow Cytometry Assay kit (Life Technologies) containing:
    • 5‐Ethynyl‐2′‐deoxyuridine (EdU, component A)
    • Alexa Fluor 488 azide in DMSO (component B)
    • DMSO (component C)
  • Alexa Fluor 647−conjugated goat anti‐mouse IgG (H+L; Invitrogen, cat. no. A‐21235)
  • Vacuum aspirator (Costar)
  • Additional reagents and equipment for analysis with BrdU using the Fenton reaction (see protocol 1)

Alternate Protocol 1: Fluorescence Immunocytochemistry and Image Acquisition using a High Content Analysis (HCA) Reader

  Additional Materials (also see protocol 1)
  • Cells (e.g., U‐2 OS)
  • Test compounds
  • Cytotoxic agents as reference compounds (e.g., camptothecin or paclitaxel, Sigma)
  • 10% (v/v) methanol‐free formaldehyde in PBS
  • 0.3% (v/v) Triton X‐100 in cold PBS
  • Primary antibody, e.g., rabbit anti‐cleaved‐caspase‐3 (Asp 175; Cell Signaling Technology, cat. no. 9661L) or rabbit anti−histone H3 (phospho Ser10; Upstate/Millipore, cat. no. 06‐570)
  • Alexa Fluor 647−conjugated goat anti‐rabbit IgG (H+L; Invitrogen, cat. no. A‐21244)
  • 0.1 M sodium ascorbate (see recipe)
  • 0.01 M Cu(II) sulfate
  • 1 mg/ml DAPI stain in water
  • 96‐well tissue culture plates: e.g., black, clear‐bottom ViewPlate‐96F (PerkinElmer Life Sciences)
  • Multichannel vacuum aspirator (type 4931, Costar)
  • 12‐well manual liquid handling or automated workstation (e.g., Beckman Coulter Biomek 2000; optional)
  • Adhesive seal (Greiner Bio‐One, cat. no. 676001)
  • HCS analyzer (e.g., ArrayScan VTI high‐content screening reader, ThermoFisher Scientific)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Bautista, P., Mohedano, A.F., Casas, J.A., Zazo, J.A., and Rodriguez, J.J. 2008. An overview of the application of Fenton oxidation to industrial wastewaters treatment. J. Chem. Technol. Biotechnol. 83:1323‐1338.
  Bors, W. and Buettner, G.R. 1997. The vitamin C radical and its reactions. In: Vitamin C in Health and Disease (L. Packer and J. Fuchs, eds), pp. 75‐94. Marcel Dekker, New York.
  Bradford, J.A., and Clarke, S.T. 2010. Dual‐pulse labeling using 5‐ethynyl‐2′‐deoxyuridine (EdU) and 5‐bromo‐2′‐deoxyuridine (BrdU) in flow cytometry. Curr. Protoc. Cytom. 55:7.38.1‐7.38.15.
  Breinbauer, R. and Kohn, M. 2003. Azide‐alkyne coupling: A powerful reaction for bioconjugate chemistry. Chembiochem 4:1147‐1149.
  Buettner, G.R. and Jurkiewicz, B.A. 1996. Catalytic metals, ascorbate and free radicals: Combinations to avoid. Radiat. Res. 145:532‐541.
  Burkitt, M.J. and Gilbert, B.C. 1990. Model studies of the iron‐catalysed Haber‐Weiss cycle and the ascorbate‐driven Fenton reaction. Free Radic. Res. Commun. 10:265‐280.
  Burrows, C.J. and Muller, J.G. 1998. Oxidative nucleobase modifications leading to strand scission. Chem. Rev. 98:1109‐1152.
  Cappella, P., Gasparri, F., Pulici, M., and Moll, J. 2008. A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry A 73:626‐636.
  Cappella, P., Giansanti, V., Pulici, M., and Gasparri, F. 2013. From “Click” to “Fenton” chemistry for 5‐bromo‐2′‐deoxyuridine determination. Cytometry A 83:989‐1000.
  Cappella, P., Gasparri, F., Pulici, M., and Moll, J. 2015. Cell proliferation method: Click chemistry based on BrdU coupling for multiplex antibody staining. Curr. Protoc. Cytom. In press.
  Darzynkiewicz, Z., and Juan, G. 1997. Analysis of DNA content and BrdU incorporation. Curr. Protoc. Cytom. 2:7.7.1‐7.7.9.
  Hong, V., Presolski, S.I., Ma, C., and Finn, M.G. 2009. Analysis and optimization of copper‐catalyzed azide‐alkyne cycloaddition for bioconjugation. Angew. Chem. Int. Ed. Engl. 48:9879‐9883.
  Jain, S.S. and Tullius, T.D. 2008. Footprinting protein‐DNA complexes using the hydroxyl radical. Nat. Protoc. 3:1092‐1100.
  Koppenol, W.H. 2001. The Haber‐Weiss cycle—70 years later. Redox Rep. 6:229‐234.
  Ligasova, A., Strunin, D., Liboska, R., Rosenberg, I., and Koberna, K. 2012. Atomic scissors: A new method of tracking the 5‐bromo‐2′‐deoxyuridine‐labeled DNA in situ. PLoS One 7:e52584.
  Miller, D.M., Buettner, G.R., and Aust, S.D. 1990. Transition metals as catalysts of “autoxidation” reactions. Free Radic. Biol. Med. 8:95‐108.
  Poot, M., Rosato, M., and Rabinovitch, P.S. 2001. Analysis of cell proliferation and cell survival by continuous BrdU labeling and multivariate flow cytometry. Curr. Protoc. Cytom. 15:7.14.1‐7.14.9.
  Salic, A. and Mitchison, T.J. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:2415‐2420.
  Sigman, D.S. 1990. Chemical nucleases. Biochemistry 29:9097‐9105.
  Ubezio, P., Lupi, M., Branduardi, D., Cappella, P., Cavallini, E., Colombo, V., Matera, G., Natoli, C., Tomasoni, D., and D'Incalci, M. 2009. Quantitative assessment of the complex dynamics of G1, S, and G2‐M checkpoint activities. Cancer Res. 69:5234‐5240.
  Zhao, H., Halicka, H.D., Li, J., Biela, E., Berniak, K., Dobrucki, J., and Darzynkiewicz, Z. 2013. DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5‐ethynyl‐2′‐deoxyuridine incorporated into DNA. Cytometry A 83:979‐988.
Key References
  Bors and Buettner, 1997. See above.
  These papers provide extensive explanation of Fenton and related reactions.
  Bautista et al., 2008. See above.
  Buettner and Jurkiewicz, 1996. See above.
  Miller et al., 1990. See above.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library