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Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining
Publication Name:
Current Protocols in Cytometry
Unit Number:
Unit 7.34
DOI:
10.1002/0471142956.cy0734s45
Online Posting Date:
July, 2008 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 (see unit 7.7). 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 an alkenyl deoxyuridine (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 preferred over traditional BrdU detection since it is more sensitive and allows multicolor and multiplex analysis in FCM and imaging. Curr. Protoc. Cytom. 58:7.34.1-7.34.13. © 2011 by John Wiley & Sons, Inc.
Keywords: BrdU azide; BMA; ethynyl deoxyuridine; click chemistry; cell cycle; multiplex analysis; chemical reporter
Table of Contents
- Introduction
- Basic Protocol: Flow Cytometry Analysis of Proliferating Cells, Protein Expression, and DNA Content
- Support Protocol: Synthesis of 5¢-BMA
- Alternate Protocol: Fluorescence Immunocytochemistry and Image Acquisition by High Content Analysis (HCA) Readers
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
Materials
Basic Protocol: Flow Cytometry Analysis of Proliferating Cells, Protein Expression, and DNA Content
Materials
- 5-Ethynyl-2¢-deoxyuridine (EdU; Berry & Associates cat. no. PY7562) dissolved in 10 mM DMSO; 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% methanol (Sigma)
- 10% methanol-free formaldehyde (PolySciences)
- PBS plus 1% FBS solution
- 0.1% Triton X-100 (Sigma) in PBS
- Antibodies:
- 0.025 mg/ml anti-BrdU monoclonal mouse antibody (clone B44, BD Biosciences)
- Alexa Fluor 647 goat anti mouse IgG (H+L), (Invitrogen cat. no. A21235)
- Cleaved Caspase 3 (Asp 175) rabbit antibody (Cell Signaling Technology cat. no. 9661L)
- Histone H3 (phosphohistone H3 Ser10) rabbit antibody (Upstate/Millipore cat. no. 06-570)
- Alexa Fluor 488 goat anti rabbit IgG (H+L), (Invitrogen cat. no. A11070)
- BrdU azide probe: 5-bromo-5¢-azido-2¢,5¢-dideoxyuridine (5¢-BMA; see
- 0.1 M copper (II) sulfate (Sigma) dissolved in water (keep at 4°C)
- 1 M (+)-sodium-l-ascorbate (Sigma) dissolved in water (store up to 2 weeks at 4°C, wrapped in aluminum foil to protect from light)
- Azide deoxyuridine (Sigma), optional
- PBST (see
- Propidium iodide staining solution (PI; see
- 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., Beckton Dickinson BD FACSCalibur)
Alternate Protocol: Fluorescence Immunocytochemistry and Image Acquisition by High Content Analysis (HCA) Readers
Additional Materials (also see Basic Protocol )
- Cells (e.g., U2-OS)
- Cytotoxic agents as reference compounds (e.g., Camptothecin or Paclitaxel, Sigma)
- 0.3% Triton X-100 in PBS
- 100 mM (+)-sodium-l-ascorbate (Sigma) dissolved in water
- 10 mM copper (II) sulfate (Sigma) dissolved in water
- 1 mg/ml DAPI staining solution dissolved in water
- 96-well, black, clear-bottom ViewPlate-96F (PerkinElmer Life Sciences)
- Cell culture incubator
- Multichannel vacuum aspirator (type 4931, Costar)
- Adhesive seal (Greiner Bio-One cat. no. 676001)
- HCS analyzer (e.g., ArrayScan high-content screening reader, Thermo Fisher)
- 12-well manual liquid handling or automated workstation (e.g., Beckman Coulter Biomek 2000), optional
Looking for materials? Suppliers Guide
Figures
-
Figure 7.34.1Overview of nucleotides used for the click chemistry coupling reaction compared to BrdU. -
Figure 7.34.2Synthesis of BMA starting from BrdU. -
Figure 7.34.3Cycloaddition of terminal ethynyl residue and BMA probe in presence of copper. The triazole product leads to BrdU coupling. Derivatized DNA is suitable for anti-BrdU monoclonal detection. -
Figure 7.34.4Assessment of BMA coupling by: (A) histogram overlay example (black histogram shows optimal dose); (B) Data plotting for MFI value for positive cells and percentage of detected positive cells (optimal condition is shown in solid squares). -
Figure 7.34.5An example of a multiplex staining as assessed in untreated (A), Camptothecin-treated (B), or Paclitaxel-treated (C) cells for 7 hr and pulsed with 10 µM EdU for 30 min. A three-parameter (protein expression, EdU-BrdU, and DNA content) analysis is shown. Percentage of positive cells for cleaved caspase-3 and phospho-histone 3 are calculated based on the squares in the dot-plot. -
Figure 7.34.6Imaging assessment by ArrayScan 4.5 of EdU-pulsed U2-OS cells of 5¢-BMA and his isomer 3¢-BMA. As unspecific control, cells are treated with azido deoxyuridine (AdU) applying click chemistry conditions. -
Figure 7.34.7Flow cytometry and imaging analysis by ArrayScan 4.5 of U2-OS cells untreated or treated with 10 mM thymidine. As unspecific control, EdU-pulsed cells are treated with azido deoxyuridine (AdU) under click chemistry conditions.
Literature Cited
| Literature Cited | |
| Bock, V.D., Hiemstra, H., and Van Maarseveen, J.H. 2006. Cu(I)-catalyzed alkyne-azide “click” cycloadditions from a mechanism and synthetic perspective. Eur. J. Org. Chem. 2006:51-68. | |
| Cristofoli, W.A., Wiebe, L.I., De Clercq, E., Andrei, G., Snoeck, R., Balzarini, J., and Knaus, E.E. 2007. 5-Alkynyl analogs of arabinouridine and 2¢-deoxyuridine: Cytostatic activity against herpes simplex virus and varicella-zoster thymidine kinase gene-transfected cells. J. Med. Chem. 50:2851-2857. | |
| Gasparri, F., Cappella, P., and Galvani, A. 2006. Multiparametric cell cycle analysis by automated microscopy. J. Biomol. Screen. 11:586-598. | |
| Hsu, T.L., Hanson, S.R., Kishikawa, K., Wang, S.K., Sawa, M., and Wong, C.H. 2007. Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc. Natl. Acad. Sci. U.S.A. 104:2614-2619. | |
| Lang, P., Yeow, K., Nichols, A., and Scheer, A. 2006. Cellular imaging in drug discovery. Nat. Rev. Drug Discov. 5:343-356. | |
| Link, A.J. and Tirrell, D.A. 2003. Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition. J. Am. Chem. Soc. 125:11164-11165. | |
| Meneni, S., Ott, I., Sergeant, C.D., Sniady, A., Gust, R., and Dembinski, R. 2007. 5-Alkynyl-2¢-deoxyuridines: Chromatography-free synthesis and cytotoxicity evaluation against human breast cancer cells. Bioorg. Med. Chem. 15:3082-3088. | |
| 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. | |
| Rostovtsev, V.V., Green, L.G., Fokin, V.V., and Sharpless, K.B. 2002. A stepwise huisgen cycloaddition process: Copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41:2596-2599. | |
| 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. | |
| Key References | |
| Breinbauer, R. and Kohn, M. 2003. Azide-alkyne coupling: A powerful reaction for bioconjugate chemistry. Chembiochem 4:1147-1149. | |
| Kolb, H.C. and Sharpless, K.B. 2003. The growing impact of click chemistry on drug discovery. Drug Discov. Today 8:1128-1137. | |
| These papers provide extensive explanation for chemical understanding of click chemistry bioconjugation and lists many important references. | |
| Internet Resources | |
| http://www.scripps.edu/chem/sharpless/click.html | |
| Sharpless' click chemistry Website. | |
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