Dual‐Pulse Labeling Using 5‐Ethynyl‐2′‐Deoxyuridine (EdU) and 5‐Bromo‐2′‐Deoxyuridine (BrdU) in Flow Cytometry

Jolene A. Bradford1, Scott T. Clarke1

1 Life Technologies, Eugene, Oregon
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.38
DOI:  10.1002/0471142956.cy0738s55
Online Posting Date:  January, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Changes in DNA replication during S‐phase can give insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation utilizes the incorporation of a thymidine analog during DNA synthesis. Incorporation of multiple analogs at different time points can further define cell cycle kinetics. Traditionally, the dual‐pulse method has been done by combining 5‐bromo‐2′‐deoxyuridine (BrdU) with iododeoxyuridine or chlorodeoxyuridine, with detection using multiple cross‐reacting BrdU antibodies. This unit presents a dual‐pulse method using the thymidine analog 5‐ethyl‐2′‐deoxyuridine (EdU), detected by click chemistry, combined with BrdU labeling and detection. No cross reactivity with incorporated EdU is observed using the BrdU antibody clone MoBU‐1. EdU detection using click chemistry does not cross‐react with incorporated BrdU. Cells are first pulsed with EdU, and then pulsed with BrdU; sequential pulses of EdU, followed by BrdU, are done without removing or washing out EdU. Curr. Protoc. Cytom. 55:7.38.1‐7.38.15. © 2011 by John Wiley & Sons, Inc.

Keywords: BrdU; EdU; S‐phase; click chemistry; dual pulse; proliferation; thymidine analog

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Cytometric Analysis of Dual Pulse EDU And BrdU, Combined with DNA Content
  • Alternate Protocol 1: Dual Pulse Using Alexa Fluor 488 Azide with Anti‐BrdU Alexa Fluor 647 Conjugate and Sytox AADvanced
  • Alternate Protocol 2: Dual Pulse Using Pacific Blue Azide with Anti‐BrdU Alexa Fluor 488 Conjugate and Fxcycle Far Red
  • Alternate Protocol 3: Dual Pulse Using Pacific Blue Azide with Anti‐BrdU Alexa Fluor 488 Conjugate and Propidium Iodide
  • Alternate Protocol 4: Dual Pulse Using Alexa Fluor 647 Azide with Anti‐BrdU Pacific Blue Conjugate and Propidium Iodide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Flow Cytometric Analysis of Dual Pulse EDU And BrdU, Combined with DNA Content

  Materials
  • Cells cultured in appropriate culture medium
  • Click‐iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen, cat. no. C35002; see recipe) containing:
    • EdU
    • Click‐iT EdU buffer additive
    • CuSO 4
    • Dye azide solution
  • BrdU solution (see recipe)
  • Trypsin ( appendix 2A)
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2A)
  • 70% (v/v) ethanol, ice cold
  • 4 M HCl (store at room temperature)
  • Phosphate/citric acid buffer, pH 7.4 (see recipe)
  • Antibody diluting buffer (see recipe), prepare fresh
  • Anti‐BrdU mouse monoclonal antibody, clone MoBU‐1, Alexa Fluor 647 conjugate (Invitrogen, cat. no. B35140)
  • FxCycle Violet stain (Invitrogen, cat. no. F10347; see recipe)
  • 37°C incubator
  • Device for counting cells, such as a hemacytometer or Countess automated cell counter (Invitrogen, cat. no. C10227)
  • 15 ml screw‐capped centrifuge tubes
  • Benchtop centrifuge
  • Vortex
  • Freezer capable of temperatures ≤−20°C
  • 5‐ml, 12 × 75–mm polystyrene tubes
  • Flow cytometer equipped with 405‐nm, 488‐nm, and 633‐nm lasers (e.g., Becton Dickinson LSRII flow cytometer or similar)
  • Additional reagents and solutions for collecting cells by trypsinization ( appendix 3B)
CAUTION: BrdU and EdU are nucleoside analogs that can be incorporated into DNA. Handle and dispose of each in compliance with all pertaining local regulations. When dissolved in DMSO, which is known to facilitate the entry of organic molecules into tissue, use additional precautions appropriate for the hazards posed by such materials.CAUTION: The hazards posed by the FxCycle Violet stain have not been fully investigated. The stain is a known mutagen and may cause sensitization by inhalation and skin contact, and is irritating to the eyes, respiratory system, and skin. Do not breathe dust. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing, safety glasses, and gloves. Avoid contact with skin and eyes. Dispose of the reagents in compliance with all pertaining local regulations.CAUTION: Anti‐BrdU antibody and Click‐iT EdU Alexa Fluor 488 Flow Cytometry component E contain sodium azide as a preservative. Sodium azide is an extremely toxic and dangerous compound, particularly when combined with acids or metals, and under acidic conditions yields highly toxic hydrazoic acid. Dispose of solutions containing sodium azide properly by diluting compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.CAUTION: DMSO, provided as a solvent, is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with all pertaining local regulations.

Alternate Protocol 1: Dual Pulse Using Alexa Fluor 488 Azide with Anti‐BrdU Alexa Fluor 647 Conjugate and Sytox AADvanced

  • SYTOX AADvanced stain (Invitrogen, cat. no. S10349; see recipe)
  • 20 mg/ml RNase A (Invitrogen, cat. no. 12091‐039)

Alternate Protocol 2: Dual Pulse Using Pacific Blue Azide with Anti‐BrdU Alexa Fluor 488 Conjugate and Fxcycle Far Red

  • Click‐iT EdU Pacific Blue Flow Cytometry Assay Kit (Invitrogen, cat. no. C10418)
  • Anti‐BrdU mouse monoclonal antibody, clone MoBU‐1, Alexa Fluor 488 conjugate (Invitrogen, cat. no. B35139)
  • FxCycle Far‐Red stain (Invitrogen, cat. no. F10348; see recipe)
  • 20 mg/ml RNase A (Invitrogen, cat. no. 12091‐039)
NOTE: The entire Click‐iT EdU Pacific Blue Flow Cytometry Assay kit is stored at 2° to 6°C and is stable for up to one year. This kit differs from the other kits only in the color of the azide component.

Alternate Protocol 3: Dual Pulse Using Pacific Blue Azide with Anti‐BrdU Alexa Fluor 488 Conjugate and Propidium Iodide

  • Click‐iT EdU Pacific Blue Flow Cytometry Assay Kit (Invitrogen, cat. no. C10418)
  • Anti‐BrdU mouse monoclonal antibody, clone MoBU‐1, Alexa Fluor 488 conjugate (Invitrogen, cat. no. B35139)
  • Propidium iodide (PI) staining solution (see recipe)
NOTE: The entire Click‐iT EdU Pacific Blue Flow Cytometry Assay Kit is stored at 2° to 6°C and is stable for up to 1 year. This kit differs from the other kits only in the color of the azide component.

Alternate Protocol 4: Dual Pulse Using Alexa Fluor 647 Azide with Anti‐BrdU Pacific Blue Conjugate and Propidium Iodide

  • Click‐iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen, cat. no. C10419)
  • Anti‐BrdU mouse monoclonal antibody, clone MoBU‐1, Pacific Blue conjugate (Invitrogen, cat. no. B35140)
  • Propidium iodide (PI) staining solution (see recipe)
NOTE: The entire Click‐iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit is stored at 2° to 6°C and is stable for up to 1 year. This kit differs from the other kits only in the color of the azide component.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 7.38.1 The structures of BrdU and EdU are very similar: BrdU contains bromine in the 5th position of the pyrimidine ring while EdU contains an alkyne group in this position of the compound.
  •   FigureFigure 7.38.2 The click reaction is a copper(I)‐catalyzed variant of the Huisgen [3+2] cycloaddition between an azide and a terminal alkyne, resulting in a covalent bond in the triazole product. In this application, the incorporated EdU contains a terminal alkyne that specifically reacts with a fluorescently labeled azide dye (Alexa Fluor 488 azide shown here as an example), to covalently label nascent DNA after the EdU pulse. A solution containing the dye‐labeled azide plus copper(II) and a reductant to convert copper(II) to copper(I) is added to the prepared cells. Because the azide and the alkyne are abiotic, they are inert until catalyzed by copper(I). The highly selective bio‐orthogonal covalent reaction occurs rapidly at room temperature. The click reaction will detect the incorporated EdU, but does not cross‐react with the incorporated BrdU.
  •   FigureFigure 7.38.3 Workflow for the dual‐pulse EdU and BrdU method.
  •   FigureFigure 7.38.4 TF‐1 myeloid progenitor cells were treated with 20 µM EdU for 1 hr before adding 10 µM BrdU to the medium for 1 hr, without removal of the EdU or washing of the cells. The cells were then fixed in ethanol and their DNA was denatured by acidification with 4 N HCl. Cells were labeled with Alexa Fluor 488 azide, anti‐BrdU antibody (clone MoBU‐1)‐Alexa Fluor 647 conjugate, and FxCycle Violet stain. Data was acquired on a Becton Dickinson LSRII flow cytometer using 405‐nm, 488‐nm, and 633‐nm lasers and analyzed with DIVA 6.1 software. Both analogs can be specifically detected. Panels A‐C show individual parameter histograms of (A) EdU detection, (B) BrdU detection, and (C) DNA content. Panels D‐F show dual‐parameter plots of (D) EdU versus BrdU, (E) DNA content versus BrdU, and (F) DNA content versus EdU.

Videos

Literature Cited

   Bakker, P.J.M., Stap, J., Tukker, J., Oven, C.H.V., Veenhof, C.H.N., and Aten, J. 1992. An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo‐ and chlorodeoxyuridine incorporated into DNA. Cytometry 12:366‐373.
   Buck, S.B., Bradford, J., Gee, K.R., Agnew, B.J., Clarke, S.T., and Salic, A. 2008. Detection of S‐phase cell cycle progression using 5‐ethynyl‐2′‐deoxyuridine incorporation with click chemistry, an alternative to using 5‐bromo‐2′‐deoxyuridine antibodies. Biotechniques 44:927‐929.
   Conboy, M.J., Karasov, A.O., and Rando, T.A. 2007. High incidence of non‐random template strand segregation and asymmetric fate determination in dividing stem cells and their progeny. PLoS Biol. 5:e102.
   Diermeier‐Daucher, S, Clarke, S.T., Hill, D., Vollmann‐Zwerenz, A., Bradford, J.A., and Brockhoff, G. 2009. Cell type specific applicability of 5‐ethynyl‐2′‐deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A 75:535‐546.
   Dolbeare, F. 1995. Bromodeoxyuridine: A diagnostic tool in biology and medicine, Part I: Historical perspectives, histochemical methods and cell kinetics. Histochem. J. 27:339‐369.
   Dolbeare, F. and Selden, J.R. 1994. Immunochemical quantitation of bromodeoxyuridine: Application to cell‐cycle kinetics. Methods Cell Biol. 41:297‐316.
   Gratzner, H.G. 1982. Monoclonal antibody to 5‐bromo‐ and 5‐iododeoxyuridine: A new reagent for detection of DNA replication. Science 218:474‐475.
   Howard, A. and Pelc, S.R. 1951. Nuclear incorporation of P32 as demonstrated by autoradiographs. Exp. Cell Res. 2:178‐187.
   Leif, R.C., Stein, J.H., and Zucker, R.M. 2004. A short history of the initial application of anti‐5‐BrdU to the detection and measurement of S phase. Cytometry A 58:45‐52.
   Maecker, H.T. and Trotter, J. 2006. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69:1037‐1042.
   Pollack, A., Terry, Nguyen T.V., and Meistrich, M.L. 1993. Flow cytometric analysis of two incorporated halogenated thymidine analogues and DNA in a mouse mammary tumor grown in vivo. Cytometry 14:168‐172.
   Roederer, M. 2001. Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry 45:194‐205.
   Rostovtsev, V.V., Green, G.L., 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.
   Rothaeusler, K. and Baumgarth, N. 2006. Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry. Cytometry A 69:249‐259.
   Rothaeusler, K. and Baumgarth, N. 2007. Assessment of cell proliferation by 5‐bromodeoxyuridine (BrdU) labeling for multicolor flow cytometry. Curr. Protoc. Cytom. 40:7.31.1‐7.31.13.
   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.
   Shankey, T.V., Rabinovitch, P.S., Bagwell, B., Bauer, K.D., Duque, R.E., Hedley, D.W., Mayall, B.H., Wheeless, L., and Cox, C. 1993. Guidelines for implementation of clinical DNA cytometry. International Society for Analytical Cytology. Cytometry 14:472‐477.
   Tornøe, C.W., Christensen, C., and Meldal, M. 2002. Peptidotriazoles on solid phase: [1,2,3]‐Triazoles by regiospecific copper(i)‐catalyzed 1,3‐dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67:3057‐3064.
   Wang, Q., Chan, T.R., Hilgraf, R., Fokin, V.V., Sharpless, K.B., and Finn M.G. 2003. Bioconjugation by copper(I)‐catalyzed azide‐alkyne [3+2] cycloaddition. J. Am. Chem. Soc. 125:3192‐3193.
   Wersto, R.P., Chrest, F.J., Leary, J.F., Morris, C., Stetler‐Stevenson, M.A., and Gabrielson, E. 2001. Doublet discrimination in DNA cell‐cycle analysis. Cytometry 46:296‐306.
Internet Resources
  http://www.scripps.edu/chem/sharpless/click.html
  Sharpless' Web site for click chemistry.
  http://www.invitrogen.com/edu
  Invitrogen EdU click chemistry Web site.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library