In Vitro Assays of Chemotaxis as a Window into Mechanisms of Toxicant‐Induced Immunomodulation

Kathryn M. Pietrosimone1, Sadikshya Bhandari1, Michael G. Lemieux1, David A. Knecht1, Michael A. Lynes1

1 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.17
DOI:  10.1002/0471140856.tx1817s58
Online Posting Date:  November, 2013
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Abstract

Dysregulated cell movement can lead to developmental abnormalities, neoplasia, and immune system disorders, and there are a variety of contexts in which xenobiotics (and biologic) effects on this movement are of interest. Many toxins and toxicants have been shown to disrupt controlled cell movement. Identification of compounds that affect cell movement is crucial to drug discovery. Drug components may have unexpected consequences with respect to cell motility, which would exclude these compounds in drug development. Finally, the development of drugs that target chemotactic pathways may be useful in the treatment of tumors, which often reprogram chemotactic pathways to become metastatic. The effects of these agents on cell movement can be measured using several different in vitro chemotactic assays. This review details the procedures of three in vitro measurements of chemotaxis: the Boyden chamber, the under‐agarose assay, and the automated, real‐time, ECIS/Taxis assay, and discusses the inferences that can be drawn from the results of such studies. Curr. Protoc. Toxicol. 58:18.17.1‐18.17.24. © 2013 by John Wiley & Sons, Inc.

Keywords: chemotaxis; immunomodulation; in vitro; mechanism; toxicant; chemokine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Boyden Chamber Assay Using a 48‐Well Chemotaxis Chamber
  • Basic Protocol 2: Under‐Agarose Real‐Time Imaging Assay
  • Basic Protocol 3: ECIS/Taxis Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Boyden Chamber Assay Using a 48‐Well Chemotaxis Chamber

  Materials
  • Chemoattractant: Stromal cell–derived factor 1α (SDF‐1α; R&D Systems, cat. no. 350‐NS‐010)
  • Incomplete RPMI medium (see recipe)
  • Jurkat T cells (ATCC, cat no. T1B‐152)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Hema 3 Stat Pack (Fisher Scientific, cat. no. 23‐123‐869)
  • Neuro Probe 48‐well chemotaxis chamber (Neuro Probe Inc., cat. no. AP48; http://www.neuroprobe.com/products/ap48.html)
  • Polycarbonate membranes, PVP‐free, 5 μm (Neuro Probe Inc., cat. no. PFB5‐50; http://www.neuroprobe.com/products/ap48.html)
  • Forceps
  • Shallow plastic containers to accommodate polycarbonate membrane
  • Glass slides (∼5 × 8–cm; Fisher Scientific)
  • Microscope

Basic Protocol 2: Under‐Agarose Real‐Time Imaging Assay

  Materials
  • SeaKem GTG Agarose (BioWhittaker Molecular Applications, cat. no. 50070)
  • Jurkat T cells (ATCC, cat no. T1B‐152)
  • Chemoattractant: Stromal cell–derived factor 1α (SDF‐1α; R&D Systems, cat. no. 350‐NS‐010)
  • Complete RPMI medium (see recipe)
  • 70% ethanol
  • 1 M HEPES buffer, cell culture grade (MP Biomedicals, cat. no. 1688449)
  • Spatula
  • 4‐chamber ibiTreat μ‐slide (ibidi, cat. no. 80426, http://ibidi.com/)
  • Cannula cutting tools (prepared as described in protocol 3)
  • 9‐in. Pasteur pipet
  • Vacuum trap and low‐vacuum source
  • Razor blades
  • 18‐mm2 glass cover slips (Fisher Scientific, cat. no. 12‐541A)
  • Forceps
  • Nikon TIE microscope (Nikon Instruments)
  • Bold Line Cage Incubator (Okolabs, cat. no. H201)
  • Andor Clara CCD camera (Andor Technologies)
  • LiveCell Stage Top Incubation Platform (Pathology Devices, http://www.pathologydevices.com/)
  • Micro‐Manager (http://valelab.ucsf.edu/~MM/MMwiki/)
  • Image‐processing application Fiji (http://fiji.sc/Fiji)
  • Stage micrometer (Edmund Optics, cat. no. 36‐121)
  • MTrackJ plugin (http://www.imagescience.org/)
  • Additional reagents and equipment for preparing agarose ( protocol 3, steps 8 to 12) and cannula cutting tool ( protocol 3, step 1).

Basic Protocol 3: ECIS/Taxis Assay

  Materials
  • 10 mM cysteine
  • SeaKem GTG Agarose (BioWhittaker Molecular Applications, cat. no. 50070)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete RPMI medium (see recipe)
  • Jurkat T cells (ATCC, cat no. T1B‐152)
  • Chemoattractant: Stromal cell–derived factor 1α (SDF‐1α; R&D Systems, cat. no. 350‐NS‐010)
  • 14‐G, 6‐in. blunt‐end cannulae (Fisher Scientific, cat. no. 290‐30)
  • 5/64‐in. drill bit
  • Handheld drill
  • Drill press
  • 1‐in. wide × 2‐in. long × 1/4‐in. thick Plexiglas
  • ECIS Ztheta 8 well array system (Applied Biophysics, http://www.biophysics.com/products‐ecis8z.php)
  • ECIS 8W Chemotaxis Electrode Array (Applied Biophysics, http://www.biophysics.com/cultureware.php)
  • 15‐ml conical polypropylene tubes (e.g., BD Falcon)
  • 0.22‐μm syringe filters
  • 5¾‐in. and 9‐in.Pasteur pipets
  • Vacuum trap
  • Low vacuum source
  • Inverted microscope
  • Computer running Microsoft Excel (optional)
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Figures

Videos

Literature Cited

Literature Cited
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