Quantifying Cellular Dynamics by Fluorescence Resonance Energy Transfer (FRET) Microscopy

Hernán E. Grecco1, Philippe I. H. Bastiaens1

1 Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.22
DOI:  10.1002/0471142301.ns0522s63
Online Posting Date:  April, 2013
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The cell is a spatially organized system whose function emerges from the complex interaction of molecular components. Such local interaction of nanometer‐sized molecules generates patterns that span throughout the cell. Those patterns, in turn, regulate the molecular interactions. Understanding such simultaneous bidirectional causation requires quantifying the spatio‐temporal progression of biochemical reactions in the context of a living cell. Due to its ability to resolve micrometer‐sized structures, biological microscopy has been instrumental to the discovery and understanding of living systems. Functional fluorescence microscopy allows a cellular dynamic topographic map of proteins to be overlaid with topological information on the causality that determines protein state. Here we describe how Förster/fluorescence resonance energy transfer (FRET) can be used to measure the phosphorylation state of proteins in the context of the cell. Curr. Protoc. Neurosci. 63:5.22.1‐5.22.14. © 2013 by John Wiley & Sons, Inc.

Keywords: quantification of cellular processes; fluorescence microscopy; Förster resonance energy transfer (FRET); acceptor photobleaching; fluorescence lifetime imaging microscopy (FLIM)

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Table of Contents

  • Introduction
  • Basic Protocol 1: FRET Microscopy on Fixed Cells
  • Basic Protocol 2: Protein Labeling with Cy3.5
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: FRET Microscopy on Fixed Cells

  • Cells of interest (e.g., MCF7, Cos7, A431)
  • Low‐background‐fluorescence CO 2‐independent medium (Life Technologies or see recipe)
  • Plasmid for mCitrine‐tagged protein (e.g., EGFR‐mCitrine, SRC‐mCitrine)
  • Transfection reagent (e.g., FuGENE 6 from Roche)
  • Serum‐free medium or 0.5% serum medium (for cells that undergo apoptosis upon serum deprivation)
  • Test substance, e.g., growth factor or hormone for stimulation, drug, inhibitor
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.4
  • 4% (w/v) formaldehyde fixative solution (see recipe)
  • Quench solution: 50 mM Tris⋅Cl (pH 8.0)/100 mM NaCl or 0.1 M hydroxylamine or 0.1 M glycine
  • 0.1% (v/v) Triton X‐100 in PBS
  • 0.1% saponin in PBS or −20°C methanol (optional)
  • Antibody (e.g., PY72 monoclonal anti‐phosphotyrosine antibody) labeled with Cy3.5 (see protocol 2)
  • 1% (w/v) bovine serum albumin (BSA, fraction V) in PBS
  • Mowiol mounting medium (see recipe)
  • Molten agarose or rubber cement (optional)
  • 6‐ and 12‐well tissue culture plates
  • Coverslips
  • Jeweler's forceps
  • Microscope slides
  • Confocal laser scanning microscope (e.g., Zeiss LSM 710, Leica SP5, Olympus FV1000), equipped with an argon laser (488 nm or 514 nm line) and a diode laser (561 nm); appropriate emission filters for mCitrine and Cy3.5 (such as Chroma ET535/30 and Chroma HQ620/60m)
  • Imaging software package (e.g., ImageJ, freely available from http://rsbweb.nih.gov/ij/)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Basic Protocol 2: Protein Labeling with Cy3.5

  • Antibody (PY72 monoclonal anti‐phosphotyrosine antibody)
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.4
  • 100 mM and 10 mM bicine, pH 8.0 (adjusted with NaOH)
  • 1 M bicine, pH 9.0 (adjusted with NaOH)
  • 100 mM citric acid, pH 2.8 (adjusted with NaOH)
  • 1 M NaCl
  • Labeling buffer: 100 mM bicine (pH 8.0)/100 mM NaCl
  • Cy3.5 monofunctional sulfoindocyanine succinimide ester (Cy3.5; GE Healthcare PA23501)
  • Dimethylformamide (DMF) dried by addition of 10 to 20 mesh 3 Å pore diameter molecular sieve dehydrate (Fluka)
  • 1 M Tris adjusted with HCl to pH 8.0
  • 1‐ml protein G or protein A HiTrap columns (Amersham Pharmacia Biotech)
  • Centricon YM30 concentrators (Amicon)
  • Biogel P6DG Econopac prepacked size‐exclusion columns (5.5 × 1.5 cm, ∼10 ml; Bio‐Rad)
  • 1 ml and 10 ml syringes with HPLC Luer‐Lok fitted tubing
  • Additional reagents and equipment for spectrophotometric protein determination ( appendix 1K) and SDS‐PAGE (Gallagher, )
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Literature Cited

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