Flow Cytometric FRET Analysis of ErbB Receptor Tyrosine Kinase Interaction

Simone Diermeier‐Daucher1, Gero Brockhoff1

1 Institute of Pathology, University of Regensburg, Regensburg, Germany
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.14
DOI:  10.1002/0471142956.cy1214s45
Online Posting Date:  July, 2008
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Abstract

The homologous and heterologous interaction of members of the epidermal growth factor (EGF)–related receptor tyrosine kinase (RTK) family (ErbB or HER family receptors) upon ligand binding is the initial key event in signal transduction by these receptors. In addition to the availability of their respective ligands, the relative expression level of the four ErbB receptors triggers receptor cross-activation, which determines signal diversification and the cells' biological response. However, the function of ErbB receptors and their ligands appears highly complex, and its impact on cell growth and proliferation of normal and tumor cells is incompletely understood. Flow cytometric fluorescence resonance energy transfer (FRET) measurements facilitate the quantitative analysis of receptor interaction. This unit delineates the cell-by-cell analysis of epidermal growth factor receptor (EGFR, ErbB1, HER1) and ErbB2 (HER2) receptor interaction in ErbB2-overexpressing BT474 and SK-BR-3 breast cancer cell lines, using a dual-laser flow cytometer. ReFlex software–based quantification of energy transfer efficiency (E) directly reflects the amount of receptor interaction. Curr. Protocol. Cytom. 45:12.14.1-12.14.19. © 2008 by John Wiley & Sons, Inc.

Keywords: receptor-tyrosine-kinases; FRET; trastuzumab; pertuzumab; ErbB2 receptor; flow cytometry; breast cancer; receptor interaction; ErbB2 homodimerization

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Assessment of ErbB2-Receptor Homodimerization
  • Support Protocol: FRET Evaluation Using ReFlex Software
  • Basic Protocol 2: Assessment of EGF-Receptor Homodimerization
  • Basic Protocol 3: Assessment of EGFR/ErbB2 Heterodimerization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assessment of ErbB2-Receptor Homodimerization

 Materials
  • SK-BR-3 breast cancer cells (ATCC #HTB-30D) grown to a subconfluent density in 75-cm2 cell culture flasks (see appendix 3B for culture of mammalian cells)
  • Complete Dulbecco's modified Eagle medium (DMEM; appendix 3B)
  • Complete DMEM containing 5% fetal bovine serum (FBS; DMEM-5; appendix 3B)
  • Trastuzumab: (also known as rhuMAb 4D5 and Herceptin) humanized monoclonal antibody acting on the ErbB2 (HER2/neu) receptor
  • Accutase solution (PAA Laboratories)
  • Cyanine 3 (Cy3; donor fluorophore)-labeled pertuzumab (also known as rhuMAb 2C4 and Omnitarg; humanized antibody targeting the ErbB2-based signaling pathway; Roche Diagnostics): label with commercially available kits (e.g., Amersham Pharmacia) according to the manufacturer's instructions
  • Cyanine 5 (Cy5; acceptor fluorophore)-labeled pertuzumab (Roche Diagnostics): label with commercially available kits (e.g., Amersham Pharmacia) according to the manufacturer's instructions
  • PBS/0.1% NaN3/0.2% BSA: phosphate-buffered saline (PBS; appendix 2A) containing 0.1% NaN3 and 0.2% bovine serum albumin (BSA); store up to 2 weeks at 4°C
  • PBS/0.1% NaN3/2% BSA: store up to 2 weeks at 4°C
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • 15-ml centrifuge tubes (e.g., 16 × 125–mm polystyrene round-bottom tubes, BD)
  • Refrigerated centrifuge, 4°C
  • Water jet pump (e.g., BRAND), optional
  • 5-ml sample tubes (e.g., 12 × 75–mm polystyrene round-bottom tubes, BD)
  • Flow cytometer with a dual-laser excitation (e.g., FACSCalibur, BD): see Table 12.14.2 for required filters
  • Acquisition software that stores data in list-mode flow cytometry standard (FCS) files (e.g., CellQuest, CellQuest Pro, FACS Diva)
  • ReFlex software (see Support Protocol)
     
    Table 12.14.2 Detection Channels on the FACSCalibur for FRET Measurement with the Cy3/Cy5 and PE/Cy5 Donor/Acceptor Dye Pair
    DyeAbsorption-maxima (nm)Emission maxima (nm)Excitation wavelength (nm)Detection channelFilter
    PE480; 565578488FL2585/42
    Cy3512; 552565; 615488FL2585/42
    FRET488FL3670LP
    Cy5625-650670~635FL4661/16

NOTE: Protect labeling solutions and labeled samples from light.

Basic Protocol 2: Assessment of EGF-Receptor Homodimerization

 Materials
  • SK-BR-3 breast cancer cells (ATCC #HTB-30D) grown to a subconfluent density in 75-cm2 cell culture flasks (see appendix 3B for culture of mammalian cells)
  • A431 epidermoid carcinoma cells (ATCC #CRL-1555) grown to a subconfluent density in 75-cm2 cell culture flasks (see appendix 3B for culture of mammalian cells)
  • Complete Dulbecco's modified Eagle medium (DMEM; appendix 3B)
  • DMEM containing 5% fetal bovine serum (FBS; complete DMEM-5; appendix 3B)
  • Accutase solution (PAA Laboratories)
  • Ligand: e.g., 5 nM (30 ng/ml) recombinant human epidermal growth factor (rhEGF)
  • 117 µg/ml phycoerythrin (PE; donor fluorophore)-labeled anti-EGFR antibody (clone EGFR.1, IgG2b; BD Biosciences)
  • Cyanine 5 (Cy5; acceptor fluorophore)-labeled anti-EGFR antibody: label clone EGFR.1 (BD Biosciences) with Cy5 antibody (see Diermeier et. al., 2005)
  • PBS/0.1% NaN3/0.2% BSA: phosphate-buffered saline (PBS; appendix 2A) containing 0.1% NaN3 and 0.2% bovine serum albumin (BSA); store up to 2 weeks at 4°C
  • PBS/0.1% NaN3/2% BSA: store up to 2 weeks at 4°C
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • 15-ml centrifuge tubes
  • Refrigerated centrifuge, 4°C
  • Water jet pump (e.g., BRAND), optional
  • 5-ml sample tubes (e.g., 12 × 75–mm polystyrene round-bottom tubes, BD)
  • Flow cytometer with a dual-laser excitation (e.g., FACSCalibur, BD)
  • Acquisition software that stores data in list-mode flow cytometry standard (FCS) files (e.g., CellQuest, CellQuest Pro, FACS Diva)
  • ReFlex software (see Support Protocol)

Basic Protocol 3: Assessment of EGFR/ErbB2 Heterodimerization

 Materials
  • SK-BR-3 breast cancer cells (ATCC #HTB-30D) grown to a subconfluent density in 75-cm2 cell culture flasks (see appendix 3B for culture of mammalian cells)
  • Complete Dulbecco's modified Eagle medium (DMEM; appendix 3B)
  • Complete DMEM containing 5% fetal bovine serum (FBS; DMEM-5; appendix 3B)
  • Therapeutic antibody (e.g., trastuzumab, also known as Herceptin)
  • Accutase solution (PAA Laboratories)
  • Cyanine 3 (Cy3; donor fluorophore)-labeled pertuzumab (also known as rhuMAb 2C4 and Omnitarg; humanized antibody targeting the HER2-based signaling pathway; Roche Diagnostics): label with commercially available kits (e.g., Amersham Pharmacia) according to the manufacturer's instructions
  • Cyanine 5 (Cy5; acceptor fluorophore)-labeled pertuzumab (Roche Diagnostics): label with commercially available kits (e.g., Amersham Pharmacia) according to the manufacturer's instructions
  • Goat anti-mouse F(ab) against the H + L chain of IgG (Dianova)
  • Unlabeled anti-EGFR antibody (clone EGFR.1, IgG2b; BD Biosciences)
  • Cy3-conjugated goat anti-mouse F(ab) against the H + L chain of IgG (Dianova)
  • PBS/0.1% NaN3/0.2% BSA
  • PBS/0.1% NaN3/2% BSA
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • 5-ml centrifuge tubes
  • Refrigerated centrifuge, 4°C
  • Water jet pump (BRAND), optional
  • 5-ml sample tubes (e.g., 12 × 75–mm polystyrene round-bottom tubes, BD)
  • Flow cytometer with a dual-laser excitation (e.g., FACSCalibur)
  • Acquisition software that stores data in list-mode flow cytometry standard (FCS) files (e.g., CellQuest, CellQuest Pro, FACS Diva)
  • ReFlex software (see Support Protocol)
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Figures

  •  FigureFigure 12.14.1 Assignment of four samples for FRET measurements. Each experiment with untreated cells requires four samples (i to iv) to correctly perform FRET calculations. The receptors (black ovals) on the cell surface are labeled with antibodies or antibody fragments conjugated to donor (open circle) or acceptor (filled circle) fluorescent dyes. Experiments with cells treated with ligand would also require the addition of an unlabeled, treated cell sample (i) and a double-labeled, treated FRET sample (iv) to the experimental setup.
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Literature Cited

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