Chemotaxis Assays for Eukaryotic Cells

Sally H. Zigmond1, Ellen F. Foxman2, Jeffrey E. Segall3

1 University of Pennsylvania, Philadelphia, Pennsylvania, 2 Stanford University Medical School, Stanford, California, 3 Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.1
DOI:  10.1002/0471143030.cb1201s00
Online Posting Date:  May, 2001
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Abstract

Chemotaxis is a complex response of a cell to an external stimulus. It involves detecting and measuring the concentration of the chemoattractant, biochemical transmission of the information, and the motility and adhesive changes associated with the response. This unit describes a number of chemotaxis assays that can be used to identify chemoattractants individually and in largeā€scale screenings, to distinguish chemotaxis from chemokinesis, and to analyze cellular behavioral and biochemical responses. Some of these assays such as the filter, under agarose, and small population assays, can be used to monitor the behavior of large groups of cells; the bridge, pipet, and upshift assays can be used to analyze the responses of single cells.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Filter Assay for Chemotaxis
  • Support Protocol 1: Calculating the Distance Cells are Expected to Move in Thick Filters in the Absence of Chemotaxis
  • Basic Protocol 2: Under‐Agarose Chemotaxis Assay
  • Basic Protocol 3: Small Population Chemotaxis Assay
  • Basic Protocol 4: Bridge Chemotaxis Assay
  • Basic Protocol 5: Pipet Chemotaxis Assay
  • Basic Protocol 6: Upshift Chemotaxis Assay
  • Support Protocol 2: Using Image Analysis Programs to Assess Chemotaxis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Filter Assay for Chemotaxis

  Materials
  • Buffer for upper and lower wells (see Critical Parameters discussion on media)
  • Chemoattractant in buffer
  • Cells of interest
  • 3.7% (w/v) formaldehyde in PBS ( appendix 2A)
  • Giemsa stain (see recipe) or Diff‐Quick (EM Science) for neutrophils; hematoxylin for lymphocytes
  • 70% and 100% isopropanol
  • 1:1 (v/v) isopropanol/Americlear
  • Americlear (Baxter Biotech)
  • Permount (EM Science)
  • 15‐µm polystyrene beads (Polysciences)
  • Chemotaxis chamber: blind‐well or multiwell chamber (Fig. ; Neuro Probe) with appropriate filters, or 24‐well tissue culture plate with filter inserts (see Critical Parameters)
  • Filter scraper (Neuro Probe; optional)
  • Microscope with 40× objective and micrometer (16× objective optional)
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.1) or cell sorter

Support Protocol 1: Calculating the Distance Cells are Expected to Move in Thick Filters in the Absence of Chemotaxis

  Materials
  • Agarose plate mixture (see recipe)
  • Migration medium (see recipe)
  • Cell suspension (107/ml) in recipemigration medium
  • Chemoattractant solution in recipemigration medium
  • Absolute methanol
  • 37% (w/v) formaldehyde
  • 0.5% (w/v) Fields stain B (Gallard‐Schlesinger)
  • 2.5% (w/v) Fields stain A (Gallard‐Schlesinger)
  • 35‐mm tissue culture dishes
  • 3‐mm hole cutter (e.g., steel punch or plastic pipet tip), sterile, with a vacuum line and a catch flask
  • Template for cutting 3‐mm wells separated by 2 mm in a single line
  • Counting grid (optional)
NOTE: All culture incubations should be 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: Under‐Agarose Chemotaxis Assay

  Materials
  • Purified agar (e.g., Noble agar, Difco)
  • Bonner's salts (see recipe)
  • Dictyostelium cells at ≤5 × 106 cells/ml
  • 17 mM Sorensen's phosphate buffer, pH 6.2 (see recipe)
  • Chemoattractant solution in recipeSorensen's phosphate buffer
  • 10‐cm petri dishes

Basic Protocol 3: Small Population Chemotaxis Assay

  Materials
  • Cells: whole blood, neutrophils in suspension, or Dictyostelium discoideum
  • 0.9% (w/v) NaCl
  • HEPES‐buffered HBSS (prepare as for recipeHBSS in appendix 2A, but replace bicarbonate with 10 mM HEPES acid) containing 0.2% (w/v) recipeBSA (for neutrophils in suspension)
  • 17 mM Sorensen's phosphate buffer, pH 6.2 (for Dictyostelium; see recipe)
  • Chemoattractant in HEPES‐buffered HBSS/1% (w/v) gelatin
  • HEPES‐buffered HBSS/1% (w/v) gelatin
  • 20 × 40–mm coverslips, unwashed (neutrophils) or acid‐washed (Dictyostelium; see recipe)
  • Glass (Neuro Probe) or Plexiglas bridge chamber (Fig. ; see Critical Parameters)
  • Microscope with 40× phase objective

Basic Protocol 4: Bridge Chemotaxis Assay

  Materials
  • Chemoattractant solution in assay buffer
  • Assay buffer: 17 mM Sorensen's phosphate buffer, pH 6.2 (see recipe), with or without CaCl 2 and MgCl 2 (for Dictyostelium), or DPBS ( appendix 22); JRH Biosciences; for MTLn3 cells)
  • Cells of interest
  • Omega dot tubing (4‐in. glass capillary tubing, 1‐mm o.d. × 0.58‐mm i.d.; A‐M Stevens)
  • Pipet puller for microinjection or neurobiology, capable of producing ∼0.1‐µm tip diameters (e.g., David Kopf Instruments, Narishige, Sutter Instruments)
  • Syringe with fine‐bore needle (e.g., 3‐in., 30‐G)
  • Micromanipulator that can be attached to a microscope stage (e.g., Leitz, Narishige)
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Figures

Videos

Literature Cited

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