Measurement of Adhesion Under Flow Conditions

Dennis F. Kucik1

1 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 9.6
DOI:  10.1002/0471143030.cb0906s43
Online Posting Date:  June, 2009
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Abstract

This unit describes the analysis of dynamic cell adhesion using a flow chamber assay. The flow chamber enables the researcher to reconstruct cell systems in the presence of shear stress to assay adhesion under well&defined forces. These assays are most commonly used to study leukocyte adhesion, either to cultured endothelial cell monolayers or to purified substrates, simulating physiological interactions of leukocytes with endothelial cells. This assay can be also be used to characterize transient adhesive events or adhesion strengthening even for cells that do not normally experience shear stress, because contact time between cells and substrates and anti&adhesive forces can be closely regulated by stopping and starting the flow. Flow chamber assays are also useful for measuring bacterial adhesion under flow. Curr. Protoc. Cell Biol. 43:9.6.1‐9.6.10. © 2009 by John Wiley & Sons, Inc.

Keywords: cell adhesion; flow chamber; leukocyte adhesion

     
 
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Table of Contents

  • Basic Protocol 1: Flow Assay for Cell Adhesion
  • Basic Protocol 2: Data Analysis
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Flow Assay for Cell Adhesion

  Materials
  • Substrate coating solutions:
  •  Fibronectin, gelatin, or other extracellular matrix proteins, either alone or in combination, for cell monolayers only
  •  10 µg/ml matrix protein, such as fibronectin or collagen, in PBS ( appendix 2A), pH 7.4, for dishes and coverslips coated with purified ligand only
  • Cultured endothelial cells (unit 1.1) growing as a confluent monolayer culture in tissue culture dish or on a coverslip of a size appropriate for the chamber used
  • 1% (w/v) BSA in PBS ( appendix 2A), pH 7.4, for dishes and coverslips coated with purified ligand only
  • PBS ( appendix 2A), pH 7.4, for dishes and coverslips coated with purified ligand only
  • 100 µg/ml poly‐L‐lysine (PLL; e.g., Sigma), for coverslips coated with purified ligand only
  • 1% (v/v) glutaraldehyde, for coverslips coated with purified ligand only
  • Leukocytes or other cells of interest
  • HBSS ( appendix 2A), room temperature and 37°C
  • 2 mM 2′,7′‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein, acetoxymethyl ester (BCECF‐AM; Molecular Probes) in DMSO
  • 35‐mm plastic tissue culture dishes for GlycoTech chamber or glass coverslips of a size appropriate for the flow chambers that require them, sterilized by autoclaving, UV light, or flaming with ethanol
  • 35‐mm tissue culture dishes or 6‐well tissue culture plates, for glass coverslips only
  • Permanent marker or diamond stylus, for dishes coated with purified ligand only
  • 25‐ml syringes
  • Programmable syringe pump
  • Flow chamber (e.g., GlycoTech) and appropriate tubing
  • Inverted phase‐contrast microscope with low‐power (∼10 to 20×) objective, fluorescence optics (recommended but not required), and stage incubator set to 37°C
  • Video camera (e.g., CCD‐300T‐RC; Dage‐MTI) and other recording equipment (e.g., VCR, television monitor, video cables)
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Figures

Videos

Literature Cited

Literature Cited
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