Cell Traction

Catherine G. Galbraith1, Michael P. Sheetz1

1 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.3
DOI:  10.1002/0471143030.cb1203s00
Online Posting Date:  May, 2001
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Abstract

Traction forces are exerted by cells on their substratum as they migrate. These forces under the entire cell or subcellular regions can be measured. This unit describes several protocols for making silicone sheets to measure traction forces under the entire cell, as well as a protocol for developing a micromachined device to measure forces under subcellular regions.

     
 
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Table of Contents

  • Basic Protocol 1: Measuring Cell Traction on Wrinkling Substrates
  • Support Protocol 1: Calibrating Microneedles
  • Alternate Protocol 1: Measuring Cell Traction on Wrinkling Substrates with an Alternative Polymer
  • Alternate Protocol 2: Measuring Cell Traction on Nonwrinkling Substrates
  • Support Protocol 2: Preparing a Modified Airbrush Apparatus
  • Basic Protocol 2: Measuring Cell Traction on Micromachined Substrates
  • Support Protocol 3: Silanizing Coverslips
  • Support Protocol 4: Preparing a Polarized Reflection Cube
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measuring Cell Traction on Wrinkling Substrates

  Materials
  • Cells of interest
  • Culture medium specific for cells being used, phenol red–free and supplemented with 20 to 50 mM HEPES
  • Dimethylpolysiloxane (60,000 or 30,000 centistokes; Sigma)
  • Phenol red–free culture medium degassed using house vacuum
  • Temperature‐control system for microscope stage (or hair dryer and controller)
  • Glass coverslips, acid washed
  • Petri dishes slightly larger than the coverslips used
  • Inverted microscope with long‐working distance‐objective or upright microscope with a multi‐immersion‐medium objective, equipped with video system
  • Micromanipulator (adjustable in three dimensions; e.g., Nanishige) with needle holder (World Precision Instruments)
  • Calibrated microneedles (see protocol 2)
  • Computer with a video‐frame capture card and image‐analysis software (optional; e.g., NIH Image, available by anonymous FTP from zippy.nimh.nih.gov)
  • Macintosh computer with Scion frame grabber running
  • Additional reagents and equipment for trypsinization of cells (unit 1.1)

Support Protocol 1: Calibrating Microneedles

  Materials
  • 25‐µm‐diameter Chromel thermocouple wire (Omega Engineering)
  • Pipet puller (David Kopf Instruments)
  • Pyrex capillary glass of various diameters and wall thicknesses (Drummond Scientific)
  • Small microscope mounted with its optical axis parallel to the bench
  • Eyepiece reticule (i.e., a calibration scale that fits into the eyepiece of the microscope; Edmund Scientific)

Alternate Protocol 1: Measuring Cell Traction on Wrinkling Substrates with an Alternative Polymer

  • Phenylmethylpolysiloxane, trimethyl terminated (Dow Corning 710 fluid)
  • Ultraviolet lamp (UVGL‐58 from UVP)

Alternate Protocol 2: Measuring Cell Traction on Nonwrinkling Substrates

  • Vacuum grease or Valap (unit 13.1)
  • Dimethylpolysiloxane (12,500 centipoise; Sigma)
  • 1‐µm liquid bead suspension (Polysciences)
  • Phenol red–free formulation of culture medium
  • Culture medium degassed using house vacuum
  • 22 × 8–mm Pyrex cylinders (Bellco Glass)
  • Glass coverslips, acid washed
  • Airbrush apparatus (see protocol 5)
  • Vacuum source
  • Sputter coater (e.g., Electron Microscopy Sciences)

Support Protocol 2: Preparing a Modified Airbrush Apparatus

  Materials
  • Nebulizer (DeVilbiss model 646)
  • Drying reservoir (16‐liter container; Nalgene)
  • T‐connector
  • Connecting tubing
  • Glass transfer pipet with tip drawn to 1‐mm‐i.d. opening
  • Impactors (37‐mm Air Sampling Cassettes; model 4339, Gelman)

Basic Protocol 2: Measuring Cell Traction on Micromachined Substrates

  Materials
  • 50% (v/v) hydrofluoric acid
  • Appropriate extracellular matrix (ECM; e.g., 40 µg/ml laminin)
  • 25% (w/v) MgSO 4
  • Phenol red–free culture medium
  • Nanofabrication facility
  • Computer‐aided drawing (CAD) software package
  • Polarized reflection cube (see protocol 7)
  • Fluorescence illumination for microscope
  • Red filter (645 nm long‐pass)
  • Cloning cylinders
  • Silanized coverslips (see protocol 7)
  • Sylgard 184 (Dow Corning)

Support Protocol 3: Silanizing Coverslips

  Materials
  • 20% HNO 3
  • Acetone, reagent grade
  • 1,1,1,3,3,3‐Hexamethyldisilazane (HMDS; Aldrich)
  • 70% ethanol
  • Glass coverslips and coverslip racks (Thomas Scientific)
  • Pyrex dish large enough to hold coverslip racks
  • Drying oven
  • Nitrogen gas cylinder with regulator
  • 3‐cc syringe with needle

Support Protocol 4: Preparing a Polarized Reflection Cube

  Materials
  • Fluorescence slider or cube (available from microscope manufacturers)
  • 50‐50 mirror (Chroma)
  • Polarizers (Chroma)
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Figures

Videos

Literature Cited

Literature Cited
   Burton, K. and Taylor, D. 1997. Traction forces of cytokinesis measured with optically modified elastic substrata. Nature 385:450‐454.
   Cramer, L., Siebert, M., and Mitchison, T. 1997. Identification of novel polarity actin filament bundles in locomoting heart fibroblasts; Implications of the generation of motile force. J. Cell Biol. 136:1287‐1305.
   Dembo, M., Oliver, T., Ishihara, A., and Jacobson, K. 1996. Imaging the traction stresses exerted by locomoting cells with the elastic substratum method. Biophys. J. 70:2008‐2022.
   Dennerll, T., Joshi, H., Steel, V., Buxbaum, R., and Heidemann, S. 1988. Tension and compression in the cytoskeleton of PC‐12 neurites II: Quantitative measurements. J. Cell Biol. 107:665‐674.
   Felder, S. and Elson, E. 1990. Mechanics of fibroblast locomotion: Quantitative analysis of forces and motions at the leading lamellas of fibroblasts. J. Cell Biol. 111:2513‐2526.
   Galbraith, C. and Sheetz, M. 1997. A new bend on measuring local traction forces: Micromachined substrate. Proc. Natl. Acad. Sci. U.S.A. 94:9114‐9118.
   Harris, A., Wild, P., and Stopak, D. 1980. Silicone rubber substrata: A new wrinkle in the study of cell locomotion. Science 208:117‐118.
   Harris, A., Stopak, D., and Wild, P. 1981. Fibroblast traction as a mechanism for collagen morphogenesis. Nature 290:249‐251.
   Lamoureux, P., Buxbaum, R., and Heidemann, S. 1989. Direct evidence that growth cones pull. Nature 340:159‐162.
   Lee, J., Leonard, M., Oliver, T., Ishihara, A., and Jacobson, K. 1994. Traction forces generated by locomoting keratocytes. J. Cell Biol. 127:1957‐1964.
   Nicklas, R. 1983. Measurements of the force produced by the mitotic spindle in anaphase. J. Cell Biol. 97:542‐548.
   Oliver, T., Lee, J., and Jacobson, K. 1994. Forces exerted by locomoting cells. Semin. Cell Biol. 5:139‐147.
   Oliver, T., Dembo, M., and Jacobson, K. 1995. Traction forces in locomoting cells. Cell Motil. Cytoskeleton 31:225‐240.
   Oliver, T., Jacobson, K., and Dembo, M. 1998. Design and use of substrata to measure traction forces exerted by cultured cells. Methods Enzymol. 298:497‐521.
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