Use of Spectral Fluorescence Resonance Energy Transfer to Detect Nitric Oxide‐Based Signaling Events in Isolated Perfused Lung

Claudette M. St. Croix1, Eileen M. Bauer2

1 Center for Biological Imaging, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 2 Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.13
DOI:  10.1002/0471142956.cy1213s45
Online Posting Date:  July, 2008
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Fluorescence resonance energy transfer (FRET) is a fluorescence microscopy technique suitable for live cells and capable of detecting changes in the conformational state of a single protein or the distance between two interacting proteins when the proteins are conjugated with appropriate donor and acceptor fluorophores. Confocal‐based spectral detection systems enable the resolution of fluorescent images by providing full spectral information for each voxel of the image without switching of optical filters. Furthermore, using calibration spectra, it is possible to unambiguously separate the cross‐talk between overlapping donor and acceptor emissions. This unit describes the use of confocal‐based spectral imaging of nitric oxide (NO) sensitive FRET reporters in the vasculature of the intact, isolated perfused mouse lung. This type of in situ imaging approach allows the visualization and study of temporal molecular signaling events within the appropriate physiologic microenvironment of the intact, living organ. Curr. Protocol. Cytom. 45:12.13.1‐12.13.12. © 2008 by John Wiley & Sons, Inc.

Keywords: nitric oxide; fluorescence resonance energy transfer; lung; confocal microscopy

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolating and Perfusing Mouse Lung
  • Basic Protocol 2: No‐Induced Protein Modifications Detected by FRET Using Spectral Confocal Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolating and Perfusing Mouse Lung

  • Lipsosomes: 1:1 molar ratio of 1,2‐dioleoyl‐3‐trimethylammonium‐propane (DOTAP)/cholesterol (see Ma et al., , b)
  • Fluorescent reporter construct: e.g., FRET‐MT adenovirus or cygnet‐2 reporter (see ), containing cyan fluorescent protein (CFP; λ ex = 440, λ em = 480) and yellow fluorescent protein (YFP; λ ex = 514, λ em = 527)
  • C57BL/6J adult male mice (e.g., The Jackson Laboratory)
  • 0.9% (w/v) NaCl
  • 20 mg/ml Avertin working solution (see recipe)
  • Heparin
  • Perfusion buffer (see recipe)
  • Mouse restrainer (e.g., see Donovan and Brown, )
  • Insulin syringe (for injecting liposomes and DNA)
  • Peristaltic pump
  • Pressure transducers
  • Premixed normal O 2 gas cylinders (5% CO 2, 21% O 2, balance N 2)
  • Scalpel, curved needle, silk sutures, sterile gauze
  • 1‐cc syringe with 26‐G needle (for injecting anesthesia)
  • 20‐G blunt metal cannula (e.g., Luer stub LS20, Instech Laboratories)
  • Blunt‐tipped surgical scissors
  • Dissecting microscope with 2× magnification
  • Curved‐tip hemostats
  • Pulmonary artery (PA) and left atrial (LA) cannulae (i.d., 1.0 mm; o.d., 1.6 mm)
  • Petri dish with coverslip bottom (e.g., Delta T dish, Bioptics)
  • Perfusate and airway lines: polyethylene PE‐50 tubing
  • Humidified environmental chamber: 6 × 6 × 2–in. Plexiglas chamber custom built in‐house to fit atop the Zeiss 510 META microscope stage; contains a small, 37°C heated water bath and ports for tracheal, pulmonary arterial, and atrial cannulae and a port with tubing for premixed gas tanks (see Fig. )
  • Spectral detection microscope system (e.g., Zeiss 510 METASystem with 40× PlanNeoFluar, 1.3‐NA, oil‐immersion objective)

Basic Protocol 2: No‐Induced Protein Modifications Detected by FRET Using Spectral Confocal Microscopy

  • Isolated perfused lung (IPL) preparation from mouse expressing a FRET reporter ( protocol 1)
  • 500 µM nitric oxide (NO) donor in perfusion buffer (see recipe): e.g., dilute with perfusion buffer from 100 mM DETA NONOate (Alexis Biochemicals) solution in H 2O, pH 8
  • Spectral detection microscope system (e.g., Zeiss 510 META with 40× PlanNeoFluar, 1.3‐NA, oil‐immersion objective) and computer with data acquisition and analysis software (e.g., Zeiss)
  • Argon laser (HFT 458, Zeiss)
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Literature Cited

Literature Cited
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