Cell Wound Assays

Paul L. McNeil1, Mark F.S. Clarke2, Katsuya Miyake3

1 Medical College of Georgia, Augusta, Georgia, 2 Universities Space Research Associates, Houston, Texas, 3 Fukushima Medical University, Fukushima, Japan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.4
DOI:  10.1002/0471143030.cb1204s02
Online Posting Date:  May, 2001
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Abstract

Many mammalian and invertebrate cells that reside in mechanically active tissue environments normally and frequently experience disruptions in plasma membrane integrity which are rapidly repaired and do not lead to cellular death. This unit describes methods for identifying “wounded” cells, often present as a minority in culture or tissues. The use of these methods can provide information about the frequency and extent of cellular wounding.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Wound Detection in Cultured Monolayers Using Fluorescein Dextran
  • Alternate Protocol 1: Wound Detection in Mammalian Tissues Using Fluorescein Dextran
  • Alternate Protocol 2: Wound Detection Using Albumin as a Wound Tracer
  • Alternate Protocol 3: Electron Microscope Visualization of Wound Tracers
  • Support Protocol 1: Quantification of Wounding Frequency and Intensity: Image Analysis
  • Support Protocol 2: Quantification of Wounding Frequency and Intensity: Flow Cytofluorometry
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Wound Detection in Cultured Monolayers Using Fluorescein Dextran

  Materials
  • Adherent cells of interest (e.g., human umbilical vein endothelial cells; HUVEC; unit 2.3)
  • Cell culture medium (e.g., DMEM/10% FBS for HUVEC; Life Technologies)
  • Dulbecco's phosphate‐buffered saline (DPBS), sterile (Life Technologies; appendix 2A)
  • 5 mg/ml FDxLys solution: lysine‐fixable fluorescein dextran, 10,000 mol. wt. (Molecular Probes) in DPBS
  • 3.7% (w/v) formaldehyde (Fisher) in DPBS
  • Antibleaching microscopic mounting medium (e.g., ProLong, Molecular Probes)
  • 22 × 22 × 1–mm glass coverslips (Fisher), sterilized by flaming with ethanol (unit 1.3)
  • Sterile petri dishes (Falcon; e.g., 100 × 15–mm with four compartments)

Alternate Protocol 1: Wound Detection in Mammalian Tissues Using Fluorescein Dextran

  Materials
  • Male Sprague‐Dawley rats (250 g)
  • Lysine‐fixable fluorescein dextran (FDxLys; Molecular Probes; 10,000 mol. wt.)
  • Dulbecco's phosphate‐buffered saline (DPBS), sterile (Life Technologies; appendix 2A)
  • 1% (w/v) procaine solution in DPBS, 37°C
  • 4% and 8% (w/v) freshly prepared formaldehyde solution in DPBS
  • 10%, 20%, and 30% (w/v) sucrose solutions in DPBS
  • Tissue‐Tek OCT compound (Fisher)
  • OCT/sucrose solution: DPBS containing 50% (v/v) OCT compound and 30% (w/v) sucrose
  • Isopentane
  • Liquid nitrogen
  • 100 mM Tris⋅Cl ( appendix 2A; but adjust to pH 7.0)
  • Antibleaching microscope mounting medium (e.g., ProLong; Molecular Probes)
  • 500‐ml reservoirs
  • 0.3‐meter lengths of 0.2‐mm‐i.d. plastic tubing
  • 1.5‐meter length of 0.1‐mm‐i.d. plastic tubing
  • Three‐way valve
  • 5‐cm‐long, 1‐ to 2‐mm‐o.d. blunt cannula
  • 18‐G needles
  • 1‐ml syringes
  • Cryostat microtome
  • Superfrost plus microscope slides (Fisher) coated with the appropriate adhesion factor (units 2.3 & 4.3)

Alternate Protocol 2: Wound Detection Using Albumin as a Wound Tracer

  • 10 mM ammonium chloride solution in DPBS
  • 0.1% (v/v) Triton X‐100 solution in DPBS
  • Wash buffer: 0.05% (v/v) Triton X‐100 in DPBS
  • H 2O 2/NaN 3/DPBS: 0.3% (v/v) hydrogen peroxide and 2% (w/v) sodium azide in DPBS (add stock H 2O 2 to azide buffer immediately before use)
  • Heat‐inactivated sheep serum (Life Technologies)
  • Horseradish peroxidase (HRP)–conjugated sheep anti–rat serum albumin (RSA) polyclonal antibody (Organon Teknika Cappel)
  • 100 mM Tris⋅Cl, pH 7.0 ( appendix 2A; but adjust pH to 7.0)
  • HRP substrate kit (Vector Laboratories) with 3,3′‐diaminobenzidine (DAB)
  • 50%, 75%, 90%, 95%, 99%, and 100% (v/v) EM‐grade ethanol
  • EM‐grade xylene
  • Permanent mounting medium (e.g., Cytoseal 60, Stephen's Scientific)
NOTE: All staining procedures are carried out at room temperature unless otherwise specified.

Alternate Protocol 3: Electron Microscope Visualization of Wound Tracers

  • 70% (v/v) EM‐grade ethanol
  • LR‐White acrylic resin, hard (EM Science)
  • Goat anti–rat serum albumin (RSA) primary antibody (Oregon Teknika Cappel)
  • Biotinylated rabbit anti–goat IgG secondary antibody (Oregon Teknika Cappel)
  • Streptavidin‐gold (10‐µm particles; Auroprobe EM kit; Amersham)
  • Intense Silver Enhancement kit (Amersham) containing silver salt (solution A), initiator (solution B), and sodium thiosulfate solution
  • Immersion oil
  • 100 mM sodium cacodylate, pH 7.4/1% (w/v) osmium tetroxide
  • 60°C oven
  • Formvar‐coated nickel grids (Monsanto)
  • Additional reagents and equipment for preparing and handling ultrathin sections and for uranyl acetate/lead citrate staining (e.g., Glauert, )
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Figures

Videos

Literature Cited

Literature Cited
   Bi, G‐.Q., Alderton, J.M., and Steinhardt, R.A. 1995. Calcium‐regulated exocytosis is required for cell membrane resealing. J. Cell Biol. 131:1747‐1758.
   Chen, W.T. 1981. Mechanism of retraction of the trailing edge during fibroblast movement. J. Cell Biol. 90:187‐200.
   Clarke, M.S.F. and McNeil, P.L. 1994. Syringe loading: A method for inserting macromolecules into cells in suspension. In Cell Biology: A Laboratory Handbook, Vol. 3. (J.E. Cells, ed.) pp. 30‐36. Academic Press, San Diego.
   Clarke, M.S.F., Caldwell, R.W., Miyake, K., and McNeil, P.L. 1995. Contraction‐induced cell wounding and release of fibroblast growth factor in heart. Circ. Res. 76:927‐934.
   Glauert, A.M. 1975. Fixation, dehydration and embedding of biological specimens. In Practical Methods in Electron Microscopy, Vol. 3 (A.M. Glauert, ed.). Elsevier Scientific Publishing, New York.
   Grembowicz, K.P., Sprague, D., and McNeil, P.L. 1999. Temporary disruption of the plasma membrane is required for c‐fos expression in response to mechanical stress. Mol. Biol. Cell. In press.
   McNeil, P.L. 1989. Incorporation of macromolecules into living cells. Methods Cell Biol. 29:153‐173.
   McNeil, P.L. 1993. Cellular and molecular adaptations to injurious mechanical force. Trends Cell Biol. 3:302‐307.
   McNeil, P.L. and Ito, S. 1990. Molecular traffic through plasma membrane disruptiuons of cells in vivo. J. Cell Sci. 96:549‐556.
   McNeil, P.L. and Khakee, R. 1992. Disruptions of muscle fiber plasma membranes. Role in exercise‐induced damage. Am. J. Pathol. 140:1097‐1109.
   McNeil, P.L. and Steinhardt, R.A. 1997. Loss, restoration and maintenance of plasma membrane integrity. J. Cell. Biol. 137:1‐4.
   McNeil, P.L., Hohman, T.C., and Muscatine, L. 1981. Mechanisms of nutritive endocytosis. II. The effect of charged agents on phagocytic recognition by digestive cells. J. Cell Sci. 52:243‐269.
   McNeil, P.L., Murphy, R.F., Lanni, F., and Taylor, D.L. 1984. A method for incorporating macromolecules into adherent cells. J. Cell Biol. 98:1556‐1564.
   Miyake, K. and McNeil, P.L. 1995. Vesicle accumulation and exocytosis at sites of plasma membrane disruption. J. Cell Biol. 131:1737‐1745.
   Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Orfao, A., Rabinovitch, P.S., Stewart, C.C., Tanke, H.J., and Wheeless, L.L. 1999. Current Protocols in Cytometry. John Wiley & Sons, New York.
   Shapiro, H.M. 1995. Practical Flow Cytometry., 3rd ed. Wiley‐Liss, New York.
   Smallheiser, N.R. 1996. Proteins in unexpected locations. Mol. Biol. Cell 7:1003‐1014.
   Steinhardt, R.A., Bi, G., and Alderton, J.M. 1994. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 263:390‐393.
   Swanson, J.A. and McNeil, P.L. 1987. Nuclear reassembly excludes large macromolecules. Science 238:548‐550.
   Terasaki, M., Miyake, K., and McNeil, P.L. 1997. Large plasma membrane disruptions are rapidly resealed by Ca2+‐dependent vesicle‐vesicle fusion events. J. Cell Biol. 139:63‐74.
   Wolff, J.A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A., and Felgner, P.L. 1990. Direct gene transfer into mouse muscle in vivo. Science 247:1465‐1468.
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