Assessment of Cell Proliferation by 5‐Bromodeoxyuridine (BrdU) Labeling for Multicolor Flow Cytometry

Kristina Rothaeusler1, Nicole Baumgarth1

1 University of California, Davis, Davis, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.31
DOI:  10.1002/0471142956.cy0731s40
Online Posting Date:  April, 2007
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Cell proliferation assays are used for a large variety of applications in the life sciences. This unit describes a flow‐cytometry‐based method that uses BrdU labeling in conjunction with multiple fluorescently labeled cell surface markers, allowing extensive phenotypic characterization of dividing cells in addition to assessment of their frequency. BrdU labeling has the advantage of constituting a nonradioactive technique that, when combined with additional fluorescent‐based reagents, avoids time‐consuming and often costly cell isolation procedures. Moreover, because BrdU is stably integrated into the DNA, it can be measured in a cell for many months. The method presented in this unit is based on paraformaldehyde fixation and reversible saponin‐based cell membrane permeabilization, which maintains cell integrity as well as fluorescent labeling with a large number of different fluorochromes, allowing 10‐ to 12‐color flow cytometric analysis of proliferating cells.

Keywords: DNA labeling; intracellular staining; FACS; turnover; proliferation

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Table of Contents

  • Basic Protocol 1: Flow Cytometric BrdU Staining Procedure Using Commercially Available Fixation and Permeabilization Reagents
  • Alternate Protocol 1: Flow Cytometric BrdU Staining Procedure Using Fixation and Permeabilization Reagents Prepared In‐House
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Flow Cytometric BrdU Staining Procedure Using Commercially Available Fixation and Permeabilization Reagents

  • 5‐bromodeoxyuridine (BrdU; Sigma‐Aldrich; store at −20°C)
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2A)
  • Mice
  • Staining medium (see recipe)
  • ACK red blood cell lysis buffer (see recipe)
  • Blocking antibody: 10 µg/ml unlabeled purified anti‐CD16/CD32 (clone 2.4.G2; eBioscience) in staining medium
  • FITC‐conjugated anti‐BrdU antibody (e.g., clone 3D4; Invitrogen)
  • Antibody conjugates for surface staining (for thymocytes, anti‐CD4‐PE/anti‐CD8‐APC; for B cells, anti‐CD19‐APC, anti‐CD45R‐PE)
  • Fluorescent live/dead discrimination stain (e.g., LIVE/DEAD Fixable Violet Dead Cell Stain Kit for flow cytometry; Invitrogen)
  • Cytofix/Cytoperm solution (BD Biosciences; or other paraformaldehyde/saponin‐based commercial solution for intracytoplasmic cytokine staining
  • Perm/Wash buffer (BD Biosciences;
  • 10% (v/v) dimethylsulfoxide (DMSO)/90% fetal bovine serum (FBS)
  • Lyophilized DNase I Code DP (Worthington)
  • Buffered saline solution (BSS; see recipe)
  • 5‐ml, 12 × 75–mm polystyrene tubes with caps
  • 50‐µm pore size nylon mesh (Fairmont Fabrics,
  • Refrigerated centrifuge and microtiter plate carrier
  • Glass microscope slides, frosted
  • 96‐well round‐bottom microtiter plates
  • Additional reagents and equipment for intraperitoneal injection of mice (Donovan and Brown, ), euthanasia of mice (Donovan and Brown, ), removal of lymphoid tissues (Reeves and Reeves, ), and counting live cells by trypan blue exclusion ( appendix 3B)
NOTE: Different reactive dyes are available that can be excited by a 488‐nm blue laser line (which is the common line on benchtop cytometers) rather than the 407‐nm violet laser line used here.CAUTION: BrdU may cause skin irritation and may be harmful if absorbed through the skin or inhaled. Work with appropriate personal protective gear.

Alternate Protocol 1: Flow Cytometric BrdU Staining Procedure Using Fixation and Permeabilization Reagents Prepared In‐House

  • Fixation solution (4% paraformaldehyde; see recipe and Sander et al., )
  • Permeabilization solution (see recipe; also see Sander et al., )
  • Wash solution (0.5% saponin; see recipe for 10% stock solution)
Carry out the protocol 1 with the following modifications at the indicated steps. Perform all other steps as described in the protocol 1.
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Literature Cited

Literature Cited
   Anderson, M.T., Baumgarth, N., Haugland, R.P., Gerstein, R.M., Tjioe, T., and Herzenberg, L.A. 1998. Pairs of violet‐light‐excited fluorochromes for flow cytometric analysis. Cytometry 33:435‐444.
   Andersson, U., Hallden, G., Persson, U., Hed, J., Moller, G., and DeLey, M. 1988. Enumeration of IFN‐gamma‐producing cells by flow cytometry: Comparison with fluorescence microscopy. J. Immunol. Methods 112:139‐142.
   Baumgarth, N. and Bigos, M. 2004. Optimization of emission optics for multicolor flow cytometry. Methods Cell. Biol. 75:3‐22.
   Baumgarth, N. and Roederer, M. 2000. A practical approach to multicolor flow cytometry for immunophenotyping. J. Immunol. Methods 243:77‐97.
   BD Biosciences. 2004. BD Biosciences Pharmingen BrdU Flow Kit Manual. BD Biosciences, Franklin Lakes, N.J.
   Bigos, M., Baumgarth, N., Jager, G.C., Herman, O.C., Nozaki, T., Stovel, R.T., Parks, D.R., and Herzenberg, L.A. 1999. Nine color eleven parameter immunophenotyping using three laser flow cytometry. Cytometry 36:36‐45.
   Carayon, P. and Bord, A. 1992. Identification of DNA‐replicating lymphocyte subsets using a new method to label the bromo‐deoxyuridine incorporated into the DNA. J. Immunol. Methods 147:225‐230.
   Chattopadhyay, P.K., Price, D.A., Harper, T.F., Betts, M.R., Yu, J., Gostick, E., Perfetto, S.P., Goepfert, P., Koup, R.A., De Rosa, S.C., Bruchez, M.P., and Roederer, M. 2006. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat. Med. 12:972‐977.
   De Rosa, S.C., Herzenberg, L.A., and Roederer, M. 2001. 11‐color, 13‐parameter flow cytometry: identification of human naive T cells by phenotype, function, and T‐cell receptor diversity. Nat. Med. 7:245‐248.
   Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
   Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Forster, I. and Rajewsky, K. 1990. The bulk of the peripheral B‐cell pool in mice is stable and not rapidly renewed from the bone marrow. Proc. Natl. Acad. Sci. U.S.A. 87:4781‐4784.
   Forster, I., Vieira, P., and Rajewsky, K. 1989. Flow cytometric analysis of cell proliferation dynamics in the B cell compartment of the mouse. Int. Immunol. 1:321‐331.
   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.
   Penit, C. and Vasseur, F. 1993. Phenotype analysis of cycling and postcycling thymocytes: Evaluation of detection methods for BrdUrd and surface proteins. Cytometry 14:757‐763.
   Reeves, J.P. and Reeves, P.A. 1991. Removal of lymphoid organs. Curr. Protoc. Immunol. 1:1.9.1‐1.9.3.
   Reome, J.B., Johnston, D.S., Helmich, B.K., Morgan, T.M., Dutton‐Swain, N., and Dutton, R.W. 2000. The effects of prolonged administration of 5‐bromodeoxyuridine on cells of the immune system. J. Immunol. 165:4226‐4230.
   Rosiepen, G., Arslan, M., Clemen, G., Nieschlag, E., and Weinbauer, G.F. 1997. Estimation of the duration of the cycle of the seminiferous epithelium in the non‐human primate Macaca mulatta using the 5‐bromodeoxyuridine technique. Cell Tissue Res. 288:365‐369.
   Rothaeusler, K. and Baumgarth, N. 2006. Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry. Cytometry A 69:249‐259.
   Sander, B., Andersson, J., and Andersson, U. 1991. Assessment of cytokines by immunofluorescence and the paraformaldehyde‐saponin procedure. Immunol. Rev. 119:65‐93.
   Schittek, B. and Rajewsky, K. 1990. Maintenance of B‐cell memory by long‐lived cells generated from proliferating precursors. Nature 346:749‐751.
   Schittek, B., Rajewsky, K., and Forster, I. 1991. Dividing cells in bone marrow and spleen incorporate bromodeoxyuridine with high efficiency. Eur. J. Immunol. 21:235‐238.
   Tough, D.F. and Sprent, J. 1996. Measurement of T and B cell turnover with BrdU. Curr. Protoc. Immunol. 18:4.7.1‐4.7.6.
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