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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Abstract

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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Materials

Basic Protocol 1: Flow Cytometric BrdU Staining Procedure Using Commercially Available Fixation and Permeabilization Reagents

  Materials
  • 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; http://www.bdbioscience.com) or other paraformaldehyde/saponin‐based commercial solution for intracytoplasmic cytokine staining
  • Perm/Wash buffer (BD Biosciences; http://www.bdbioscience.com)
  • 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, http://www.fairmontfabrics.com)
  • 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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Figures

Videos

Literature Cited

Literature Cited
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   BD Biosciences. 2004. BD Biosciences Pharmingen BrdU Flow Kit Manual. BD Biosciences, Franklin Lakes, N.J.
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   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.
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