In Vivo Imaging Using Quantum Dot–Conjugated Probes

Diane S. Lidke1, Peter Nagy2, Donna J. Arndt‐Jovin3

1 University of New Mexico, Albuquerque, New Mexico, 2 University of Debrecen, Debrecen, Hungary, 3 Max Planck Institute for Biophysical Chemistry, Goettingen, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 25.1
DOI:  10.1002/0471143030.cb2501s36
Online Posting Date:  September, 2007
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Abstract

This unit describes the use of quantum dots (QDs) for live‐cell imaging and the use of QDs in flow cytometry for quantitative analysis of ligand binding constants and receptor density. Conventional fluorophores and visible fluorescent protein (VFP) constructs have allowed visualization of many cellular processes. However, organic and biomolecular fluorophores have limitations in their applications, due to their small Stokes' shift and tendency to photobleach during prolonged imaging. QDs have many advantages over conventional fluorophores, including high brightness and photostability, which make them an exceptional tool for live‐cell imaging. There are a large variety of commercially available QDs with different surface reactivities and characteristics. The authors have limited the laboratory protocols presented here to the use of streptavidin‐coupled QDs because this gives almost universal applicability to any cell surface receptor by coupling the ligand or antibody that recognizes the receptor to biotin and visualizing the complex by use of QDs. Curr. Protoc. Cell Biol. 36:25.1.1‐25.1.18. © 2007 by John Wiley & Sons, Inc.

Keywords: quantum dots; live‐cell imaging; binding constants; receptor number; biotinylated ligands

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: In Vivo, In Situ Labeling of Cells with QD‐Ligand Conjugates for Fluorescence Microscopic Imaging
  • Alternate Protocol 1: Labeling Cell Surface Receptors in a Two‐Step Procedure
  • Support Protocol 1: Coupling Monobiotinylated Ligands to Streptavidin‐QDs
  • Support Protocol 2: Preparation of Preformed Complexes of QDs with Ligands with Multiple Biotins
  • Basic Protocol 2: Flow Cytometric Determination of the Binding Constants of Ligands or Antibodies
  • Basic Protocol 3: Flow Cytometric Quantitation of the Number of Antibody Binding Sites
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vivo, In Situ Labeling of Cells with QD‐Ligand Conjugates for Fluorescence Microscopic Imaging

  Materials
  • Logarithmically growing cells (unit 1.1)
  • Trypsin
  • Cell culture medium appropriate for the particular cell line
  • Tyrode's buffer plus (see recipe)
  • Preformed QD‐ligands ( protocol 3 or protocol 42)
  • LabTek 8‐well coverslip chambers (Nunc)
  • Emission filters for specific QD emission wavelengths (Chroma or Omega, also available from the microscope manufacturers)
  • Additional reagents and equipment for culture of cells (unit 1.1)

Alternate Protocol 1: Labeling Cell Surface Receptors in a Two‐Step Procedure

  Materials
  • Cells cultured in chambers or on coverslips ( protocol 1)
  • Tyrode's buffer plus (see recipe), cold
  • Bio‐ligand
  • Streptavidin‐conjugated Quantum Dots (SAvQD; Invitrogen)
  • 4% (w/v) paraformaldehyde
  • Tris‐saline buffer
  • Phosphate‐buffered saline (PBS; see recipe)
  • 10° to 12°C water bath

Support Protocol 1: Coupling Monobiotinylated Ligands to Streptavidin‐QDs

  Materials
  • Streptavidin‐conjugated Quantum Dots (SAvQD; Invitrogen)
  • Phosphate‐buffered saline (PBS)/1% (w/v) BSA (see recipe)
  • Biotinylated‐Ligand (bio‐ligand)
  • PBS (see recipe)
  • 1% (w/v) BSA
  • Shaker
  • Gel filtration spin columns, NAC‐5

Support Protocol 2: Preparation of Preformed Complexes of QDs with Ligands with Multiple Biotins

  Materials
  • Streptavidin‐conjugated Quantum Dots (SAvQD; Invitrogen)
  • PBS/1% (w/v) BSA (see recipe)
  • Bio‐ligand

Basic Protocol 2: Flow Cytometric Determination of the Binding Constants of Ligands or Antibodies

  Materials
  • Biotin‐coupled ligand or antibody
  • Avidin‐coated QD (Invitrogen or Evident Technologies) suitable for excitation with the flow cytometer available
  • Trypsin
  • Phosphate‐buffered saline (PBS)/1% (w/v) BSA (see recipe)
  • Flow cytometry tube
  • Flow cytometer
  • Flow cytometric analysis software
  • Additional reagents and equipment for coupling the ligand or antibody of interest to QDs ( protocol 3) and culture of cells (unit 1.1)

Basic Protocol 3: Flow Cytometric Quantitation of the Number of Antibody Binding Sites

  Materials
  • Cells being investigated
  • Primary unlabeled antibody
  • Secondary QD‐conjugated antibody against the primary antibody (Quantum Dot Corporation or Evident Technologies)
  • Phosphate‐buffered saline (PBS)/1% (w/v) BSA (see recipe)
  • Qifikit calibration kit (Dako)
  • Vortex
  • Qifikit calibration beads (Dako‐Cytomation, www.dako.com) or Quantum Simply Cellular beads (Bangs Laboratories, www.bangslabs.com)
  • Flow cytometer
  • Quantum Simply Cellular kit (Bangs Laboratories)
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Figures

Videos

Literature Cited

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