Ultraviolet‐Induced Detection of Halogenated Pyrimidines (UVID)

Hans‐Joerg Hammers1, Peter Schlenke1

1 University of Luebeck, Leubeck, Germany
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.15
DOI:  10.1002/0471142956.cy0715s16
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

When halogenated pyrimidines such as BrdU are used to identify DNA‐synthesizing cells, their detection with monoclonal antibodies requires DNA denaturation, which can lead to loss of cells and/or cell markers or antigens. The authors present an alternative method employing ultraviolet light to partially photolyze BrdU and induce extensive damage in the nuclei which have incorporated the pyrimidine. Under appropriate conditions the BrdU in the unfolding chromatin is detected by certain monoclonal antibodies. Since no denaturation step or enzymatic treatment is required, this method preserves cellular markers and avoids enzyme‐specific artifacts. Directions are given for both coagulative (ethanol) and crosslinking (formaldehyde) fixation. Keywords: flow cytometry; BrdU; UV; photolysis; hypotonic; cell cycle; proliferation When halogenated pyrimidines such as BrdU are used to identify DNA‐synthesizing cells, their detection with monoclonal antibod

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

Table of Contents

  • Basic Protocol 1: Ultraviolet‐Induced Detection of BrdU in Ethanol‐ or Paraformaldehyde‐Fixed Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Ultraviolet‐Induced Detection of BrdU in Ethanol‐ or Paraformaldehyde‐Fixed Cells

  Materials
  • 10 mM BrdU stock solution (see recipe)
  • Exponentially growing cells
  • Phosphate buffered saline (PBS; appendix 2A) with and without 0.1% BSA
  • 70% (v/v) high purity ethanol, −20°C
  • 0.2% (w/v) paraformaldehyde in PBS, 20°C (see recipe)
  • Sodium tetraborate solution (see recipe)
  • Anti‐BrdU FITC antibody
  • 7‐AAD solution (optional; see recipe)
  • 37°C, 5% CO 2 incubator
  • Tabletop centrifuge and swinging bucket rotor
  • Hand‐held 8‐W UV‐B lamp with an intensity of ∼22 W/m2 measured 0.5 cm above the filter surface
  • 14‐ml clear polypropylene tubes (e.g., Sarstedt)
  • Clear tape or rubber band
  • Microcentrifuge
  • Vortex mixer
  • Tubes suitable for use with flow cytometer
  • Distilled water
  • Flow cytometer equipped with a 488‐nm argon laser and filters for detection of FITC (525 nm), phycoerythrin (PE; 575 nm), and 7‐AAD (650‐nm long‐pass or 675‐nm band‐pass)
NOTE: Not every clone can be used with the UVID approach. The authors recommend the Bu20a clone (Dako), but the 3D4 clone (PharMingen) and the B44 clone (Becton Dickinson) are acceptable as well.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   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.
   Dolbeare, F. 1995a. Bromodeoxyuridine: A diagnostic tool in biology and medicine. Part I: Historical perspectives, histochemical methods and cell kinetics. Histochem. J. 27:339‐369.
   Dolbeare, F. 1995b. Bromodeoxyuridine: A diagnostic tool in biology and medicine. Part II: Oncology, chemotherapy and carcinogenesis. Histochem. J. 27:923‐964.
   Dolbeare, F. 1996. Bromodeoxyuridine: A diagnostic tool in biology and medicine. Part III: Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridization. Histochem. J. 28:531‐575.
   Hammers, H.J., Kirchner, H., and Schlenke, P. 2000. Ultraviolet‐induced detection of halogenated pyrimidines: Simultaneous analysis of DNA replication and cellular markers. Cytometry 40:327‐335.
   Li, X., Traganos, F., Melamed, M.R., and Darzynkiewicz, Z. 1994a. Detection of 5‐bromo‐2‐deoxyuridine incorporated into DNA by labeling strand breaks induced by photolysis (SBIP). Int. J. Oncol. 4:1157‐1161.
   Li, X., Traganos, F., and Darzynkiewicz, Z. 1994b. Simultaneous analysis of DNA replication and apoptosis during treatment of HL‐60 cells with camptothecin and hyperthermia and mitogen stimulation of human lymphocytes. Cancer Res. 54:4289‐4293.
   Li, X., Melamed, M.R., and Darzynkiewicz, Z. 1996. Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp. Cell Res. 222:28‐37.
   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.
   Takagi, S., McFadden, M.L., Humphreys, R.E., Woda, B.A., and Sairenji, T. 1993. Detection of 5‐bromo‐2‐deoxyuridine (BrdUrd) incorporation with monoclonal anti‐BrdUrd antibody after deoxyribonuclease treatment. Cytometry 14:640‐648.
Key Reference
   Hammers et al., 2000. See above.
  This article describes the original work and gives examples of how to combine the method with the simultaneous detection of surface markers.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library