Optical Microscopy–Based Migration Assay for Human Neutrophils

Lynda M. Pierini1, Frederick R. Maxfield1

1 Weill Medical College of Cornell University, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.6
DOI:  10.1002/0471143030.cb1206s17
Online Posting Date:  February, 2003
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Abstract

This unit describes an in vitro microscopy assay for examining the migration of human neutrophils in two dimensions to identify the underlying cause of a migration defect and to evaluate a variety of migration parameters that cannot be studied using migration through a porous filter. Freshly isolated human neutrophils a placed in the chamber, stimulated, and images are collected at various time points. The data can be used to determine the effects of a pharmacological treatment on the migration of individual cells.

     
 
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Table of Contents

  • Basic Protocol 1: Acquiring Time‐Lapse Images of Migrating Neutrophils
  • Support Protocol 1: Isolating Human Neutrophils
  • Support Protocol 2: Preparing Coverslips and Microscopy Chambers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Acquiring Time‐Lapse Images of Migrating Neutrophils

  Materials
  • HBS, pH 7.4 (see recipe)
  • 50× (0.5 M) glucose stock solution (filter sterilize and store at 4°C)
  • Human neutrophils, freshly isolated (see protocol 2)
  • Formyl‐Met‐Leu‐Phe (fMLF)
  • 1.5‐ml polypropylene microcentrifuge tubes
  • End‐over‐end rotator or platform rocker
  • Air‐curtain heater (Arenberg Sage)
  • 35‐mm fibronectin‐coated coverslip dishes (see protocol 3)
  • Humidified slide warmer (LabLine Instruments)
  • Inverted microscope equipped for digital imaging with 20×, 40×, or 63× objective
NOTE: Neutrophils are readily activated by shear forces or by contact with certain surfaces. Cells should be handled gently, transferred using wide‐bore pipet tips, and kept in polypropylene (as opposed to glass or polystyrene) vessels.

Support Protocol 1: Isolating Human Neutrophils

  Materials
  • Sodium heparin
  • Healthy volunteer blood donor
  • Polymorphprep density gradient medium (Accurate Chemical & Scientific Corp.)
  • 5× PBS (see recipe)
  • HBS/glucose: HBS, pH 7.4 (see recipe), containing 10 mM glucose added from 0.5 M stock (see protocol 1)
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Tourniquet
  • Alcohol wipes
  • 21‐G needles
  • 10‐ml disposable syringes
  • Tabletop centrifuge with swinging‐bucket rotor
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.1)
NOTE: Use PBS without calcium or magnesium throughout the isolation procedure until the final resuspension step. Divalent cations in HBS will cause cells to aggregate.

Support Protocol 2: Preparing Coverslips and Microscopy Chambers

  Materials
  • Nochromix glass cleaning reagent (Godax Laboratories) mixed with H 2SO 4 per manufacturer's instructions
  • Paraffin
  • Petroleum jelly
  • 1× PBS (see recipe)
  • 0.1 mg/ml fibronectin (see recipe)
  • Glass coverslips (thickness should be matched with recommendation of microscope manufacturer)
  • 35‐mm tissue culture dishes
  • Sheet metal hole punch
  • Cotton‐tipped applicator or fine‐tipped paint brush
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Figures

Videos

Literature Cited

Literature Cited
   Allan, R. and Wilkinson, P. 1978. A visual analysis of chemotactic and chemokinetic locomotion of human neutrophil leucocytes. Use of a new chemotaxis assay with Candida albicans as gradient source. Exp. Cell Res. 111:191‐203.
   Boyden, S.V. 1962. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exp. Med. 115:453‐466.
   Dixon, H.M. and McCutcheon, M. 1936. Chemotropism of leukocytes in relation to their rate of locomotion. Proc. Soc. Exp. Biol. Med. 34:173‐176.
   Dunn, G.A. 1981. Chemotaxis as a form of directed cell behaviour: Some theoretical considerations. In Biology of the Chemotactic Response (J.M. Lackie and P.C. Wilkinson, eds.) p.1‐26. Cambridge University Press, Cambridge, U.K.
   Eddy, R., Pierini, L., Matsumura, F. and Maxfield, F. 2000. Ca2+‐dependent myosin II activation is required for uropod retraction during neutrophil migration. J. Cell Sci. 113:1287‐1298.
   Graham, I.L. and Brown, E.J. 1991. Extracellular calcium results in a conformational change in Mac‐1 (CD11b/CD18) on neutrophils. Differentiation of adhesion and phagocytosis functions of Mac‐1. J. Immunol. 146:685‐691.
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   Haston, W. and Wilkinson, P. 1988. Locomotion and chemotaxis of leukocytes: Gradient perception and locomotor capacity. Curr. Opin. Immunol. 1:5‐9.
   Hendey, B. and Maxfield, F.R. 1993. Regulation of neutrophil motility and adhesion by intracellular calcium transients. Blood Cells. 19:143‐164.
   Keller, H., Wilkinson, P., Abercrombie, M., Becker, E., Hirsch, J., Miller, M., Ramsey, W. and Zigmond, S. 1977a. A proposal for the definition of terms related to locomotion of leucocytes and other cells. Cell Biol. Int. Rep. 1:391‐397.
   Keller, H., Wilkinson, P., Abercrombie, M., Becker, E., Hirsch, J., Miller, M., Ramsey, W., and Zigmond, S. 1977b. A proposal for the definition of terms related to locomotion of leukocytes and other cells. J. Immunol. 118:1912‐1914.
   Keller, H., Wilkinson, P., Abercrombie, M., Becker, E., Hirsch, J., Miller, M., Scottramsey, W., and Zigmond, S. 1977c. A proposal for the definition of terms related to locomotion of leucocytes and other cells. Clin. Exp. Immunol. 27:377‐380.
   Mandeville, J., Ghosh, R. and Maxfield, F. 1995. Intracellular calcium levels correlate with speed and persistent forward motion in migrating neutrophils. Biophys. J. 68:1207‐1217.
   Mandeville, J., Lawson, M., and Maxfield, F. 1997. Dynamic imaging of neutrophil migration in three dimensions: Mechanical interactions between cells and matrix. J. Leukocyte Biol. 61:188‐200.
   Marks, P.W., Hendey, B., and Maxfield, F.R. 1991. Attachment to fibronectin or vitronectin makes human neutrophil migration sensitive to alterations in cytosolic free calcium concentration. J. Cell Biol. 112:149‐158.
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   Palecek, S., Loftus, J., Ginsberg, M., Lauffenburger, D., and Horwitz, A. 1997. Integrin‐ligand binding properties govern cell migration speed through cell‐substratum adhesiveness. Nature 385:537‐540.
   Pierini, L., Lawson, M., Eddy, R., Hendey, B., and Maxfield, F. 2000. Oriented endocytic recycling of alpha5beta1 in motile neutrophils. Blood 95:2471‐2480.
   Pytowski, B., Maxfield, F., and Michl, J. 1990. Fc and C3bi receptors and the differentiation antigen BH2‐Ag are randomly distributed in the plasma membrane of locomoting neutrophils. J. Cell Biol. 110:661‐668.
   Seveau, S., Eddy, R., Maxfield, F., and Pierini, L. 2001. Cytoskeleton‐dependent membrane domain segregation during neutrophil polarization. Mol. Biol. Cell. 12:3550‐3562.
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