Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

Paolo Cappella1, Fabio Gasparri1, Maurizio Pulici2, Jürgen Moll1

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 7.34
DOI:  10.1002/0471142956.cy0734s72
Online Posting Date:  April, 2015
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Abstract

Determination of incorporation of the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S‐phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5‐ethynyl‐2′‐deoxyuridine (EdU) which is incorporated into DNA. The nucleotide‐exposed ethynyl residue was then derivatized by a copper‐catalyzed cycloaddition reaction (“click chemistry” coupling) using a BrdU azide probe. The resulting DNA‐bound bromouracil moieties were then detected by commercial anti‐BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image‐based cytometry. © 2015 by John Wiley & Sons, Inc.

Keywords: BrdU azide; BMA; ethynyl deoxyuridine; click chemistry; cell cycle; multiplex analysis; chemical reporter

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Cytometry Analysis of Proliferating Cells, Protein Expression, and DNA Content
  • Alternate Protocol 1: Flow Cytometry Analysis of Proliferating Cells In Vivo for Bone Marrow and Tumor Studies
  • Alternate Protocol 2: Fluorescence Immunocytochemistry and Image Acquisition by High‐Content Analysis (HCA) Readers
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Flow Cytometry Analysis of Proliferating Cells, Protein Expression, and DNA Content

  Materials
  • 5‐Ethynyl‐2′‐deoxyuridine (EdU) dissolved in dimethyl sulfoxide (DMSO; 10 mM; cat. no. PY 7562, Berry & Associates)
  • 5‐Ethynyl‐2′‐deoxycytidine (EdC) dissolved in DMSO (10 mM; cat. no. T511307, Sigma‐Aldrich)
  • (2′S)‐2′‐deoxy‐2′‐fluoro‐5‐ethynyluridine (F‐ara‐EdU) dissolved in DMSO (40 mM; cat. no. T511293, Sigma‐Aldrich)
  • Cells cultured in appropriate culture medium (e.g., U‐2 OS, HL60)
  • Phosphate‐buffered saline (PBS) without calcium and magnesium (Invitrogen)
  • 0.25% trypsin/0.02% EDTA solution, 37°C (Invitrogen)
  • Serum‐containing medium
  • Methanol, anhydrous (cat. no. 322415, Sigma‐Aldrich)
  • 10% methanol‐free formaldehyde (PolySciences)
  • PBS plus 1% fetal bovine serum (FBS) solution
  • PBS containing 0.1% Triton X‐100 (PBST; Sigma‐Aldrich)
  • Antibodies:
  • 0.025 mg/ml anti‐BrdU monoclonal mouse antibody (clone B44, BD Biosciences)
  • Alexa Fluor 647 goat anti‐mouse IgG (H + L; cat. no. A21235, Invitrogen)
  • Cleaved caspase‐3 (Asp 175) rabbit antibody (cat. no. 9661; Cell Signaling Technology)
  • Histone H3 (phospho‐histone H3 Ser10) rabbit antibody (cat. no. 06‐570, EMD Millipore)
  • Alexa Fluor 488 goat anti‐rabbit IgG (H + L; cat. no. A11070, Invitrogen)
  • BrdU azide probe:
  • 5‐bromo‐5′‐azido‐2′,5′‐dideoxyuridine (5′‐BMA, dissolve in DMSO as 10 mM stock solution; see Cappella et al., )
  • 5‐bromo‐3′‐azido‐2′,3′‐dideoxyuridine (3′‐BMA, dissolve in DMSO as 10 mM stock solution; cat no. PY 7286, Berry & Associates)
  • 5‐bromo‐3′,5′‐diazido‐2′,3′,5′‐trideoxyuridine (3′,5′‐BDA, dissolve in DMSO as 10 mM stock solution; see Cappella et al., )
  • 0.1 M copper(II) sulfate, dissolved in water (maintain at 4°C; Sigma‐Aldrich)
  • 1 M (+)‐sodium‐L‐ascorbate, dissolved in water (Sigma‐Aldrich) or 0.5 M (+)‐sodium‐L‐ascorbate, prepared fresh from L‐ascorbic acid solution (store up to 1 week at 4°C, wrapped in aluminum foil to protect from light; see recipe)
  • 1 M L‐ascorbic acid (cat. no. A5960, Sigma‐Aldrich) dissolved in water (prepare daily and store at room temperature, wrapped in aluminum foil to protect from light)
  • Azide deoxyuridine (optional, Sigma‐Aldrich)
  • PBST (see recipe)
  • Propidium iodide staining solution (PI; see recipe)
  • 37°C incubator
  • 15‐ml screw‐capped centrifuge tubes
  • Coulter counter
  • Shaker
  • Vacuum aspirator (Costar)
  • 5‐ml (12 × 75‐mm) polystyrene tubes (BD Falcon)
  • Flow cytometer equipped with a dual laser He‐Ne for red excitation and argon excitation at 488 nm (e.g., BD FACSCalibur, Becton Dickinson)

Alternate Protocol 1: Flow Cytometry Analysis of Proliferating Cells In Vivo for Bone Marrow and Tumor Studies

  Additional Materials protocol 1Basic Protocol
  • BALB/c Nu/Nu xenograft mice (Harlan Laboratories)
  • 5‐Ethynyl‐2′‐deoxyuridine (EdU; cat. no. PY 7562, Berry Associates)
  • 70% methanol
  • Pepsin (P7000, Sigma‐Aldrich)
  • 37% HCl (∼12 M)
  • Dimethyl sulfoxide (DMSO)
  • 0.9% NaCl solution
  • 1% BSA
  • Alexa Fluor 488‐conjugated goat anti‐mouse IgG secondary antibody
  • 19‐, 21‐, and 26‐gauge needles
  • Surgical scissors
  • 35‐μm Falcon cell stainer
  • Scalpel blades
  • Additional reagents and equipment for euthanizing mice (Donovan and Brown, )

Alternate Protocol 2: Fluorescence Immunocytochemistry and Image Acquisition by High‐Content Analysis (HCA) Readers

  Additional Materials protocol 1Basic Protocol
  • Cells (e.g., U‐2 OS)
  • Cytotoxic agents as reference compounds (e.g., camptothecin or paclitaxel, Sigma‐Aldrich)
  • 0.3% Triton X‐100 in PBS
  • 100 mM (+)‐sodium‐L‐ascorbate, dissolved in water (Sigma‐Aldrich)
  • 10 mM copper(II) sulfate, dissolved in water (Sigma‐Aldrich)
  • 1 mg/ml DAPI staining solution, dissolved in water (cat. no. 62247, Pierce Biotechnology)
  • 96‐well black clear‐bottom ViewPlate‐96 F (PerkinElmer Life Sciences)
  • Multichannel vacuum aspirator (type 4931, Costar)
  • Adhesive seal (cat. no. 676001, Greiner Bio‐One)
  • High‐content screening (HCS) analyzer (e.g., ArrayScan high‐content screening reader, Thermo Scientific)
  • 8/12‐well manual pipettor or automated liquid handling workstation, optional (e.g., Beckman Coulter Biomek, 2000)
CAUTION: Formaldehyde is highly volatile, toxic, and carcinogenic. It irritates skin, eyes, mucous membranes, and the respiratory tract. Avoid breathing in vapors and use in a fume hood.
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Figures

Videos

Literature Cited

Literature Cited
  Cappella, P. and Gasparri, F. 2014. Highly multiplexed phenotypic imaging for cell proliferation studies. J. Biomol. Screen 19:145‐157.
  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 73A: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 83A:989‐1000.
  Darzynkiewicz, Z. and Juan, G. 1997. Analysis of DNA content and BrdU incorporation. Curr. Protoc. Cytom. 2:7.7.1‐7.7.9.
  Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Gasparri, F., Cappella, P., and Galvani, A. 2006. Multiparametric cell cycle analysis by automated microscopy. J. Biomol. Screen. 11:586‐598.
  Guan, L., van der Heijden, G.W., Bortvin, A., and Greenberg, M.M. 2011. Intracellular detection of cytosine incorporation in genomic DNA by using 5‐ethynyl‐2′‐deoxycytidine. ChemBioChem 12:2184‐2190.
  Hamelik, R.M. and Krishan, A. 2009. Click‐iT assay with improved DNA distribution histograms. Cytometry A 75A:862‐865.
  Hammers, H.‐J. and Schlenke, P. 2001. Ultraviolet‐induced detection of halogenated pyrimidines (UVID). Curr. Protoc. Cytom. 16:7.15.1‐7.15.6.
  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.
  Huisgen, R. 1961. Centenary lecture‐1,3‐dipolar cycloadditions. Proc. Chem. Soc. 357.
  Juan, G., Traganos, F., James, W.M., Ray, J.M., Roberge, M., Sauve, D.M., Anderson, H., and Darzynkiewicz, Z. 1998. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 32:71‐77.
  Li, X., Traganos, F., Melamed, M.R., and Darzynkiewicz, Z. 1995. Single step procedure for DNA strand‐breaks labeling. Detection of apoptosis and DNA replication. Cytometry 20:172‐180.
  Liboska, R., Ligasová, A., Strunin, D., Rosenberg, I., and Koberna, K. 2012. Most anti‐BrdU antibodies react with 2′‐deoxy‐5‐ethynyluridine – the method for the effective suppression of this cross‐reactivity. PLoS One 7:e51679.
  Ligasová, 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.
  Neef, A.B. and Luedtke, N.W. 2011. Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proc. Natl. Acad. Sci. U.S.A. 108:20404‐20409.
  Pochet, S., Dugue, L., Labesse, G., Delepierre, M., and Munier‐Lehmann, H. 2003. Comparative study of purine and pyrimidine nucleoside analogues acting on the thymidylate kinases of Mycobacterium tuberculosis and of humans. ChemBioChem 4:742‐747.
  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.
  Qu, D., Wang, G., Wang, Z., Zhou, L., Chi, W., Cong, S., Ren, X., Liang, P., and Zhang, B. 2011. 5‐Ethynyl‐2′‐deoxycytidine as a new agent for DNA labeling: Detection of proliferating cells. Anal. Biochem. 417:112‐121.
  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.
  Tang, X., Falls, D.L., Li, X., Lane, T., and Luskin, M.B. 2007. Antigen‐retrieval procedure for bromodeoxyuridine immunolabeling with concurrent labeling of nuclear DNA and antigens damaged by HCl pretreatment. J. Neurosci. 27:5837‐5844.
  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 83A:979‐988.
Key References
  Breinbauer, R. and Kohn, M. 2003. Azide‐alkyne coupling: A powerful reaction for bioconjugate chemistry. ChemBioChem 4:1147‐1149.
  These articles provide extensive explanation for chemical understanding of click‐chemistry bioconjugation and list many important references.
  Presolski, S.I., Hong, V.P., and Finn, M.G. 2011. Copper‐catalyzed azide‐alkyne click chemistry for bioconjugation. Curr. Protoc. Chem. Biol. 3:153‐162.
  Kolb, H.C. and Sharpless, K.B. 2003. The growing impact of click chemistry on drug discovery. Drug Discov. Today 8:1128‐1137.
Internet Resources
  http://www.scripps.edu/chem/sharpless/click.html
  The Sharpless Lab's click chemistry website.
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