Drug Testing in Cellular Chemotaxis Assays

Mario Mellado1, Carlos Martínez‐A1, José Miguel Rodríguez‐Frade1

1 Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Madrid, Spain
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.11
DOI:  10.1002/0471141755.ph1211s41
Online Posting Date:  June, 2008
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Abstract

Described in this unit are methods for measuring the cell migration process. While cell adhesion protocols allow study of migrating cell interactions with the endothelial matrix, cellular migration assays permit analysis of directed cell movement towards a chemotactic gradient, both in vivo and in vitro. An in vitro cell invasion protocol is provided for analysis of the sum of the cell adhesion, migration, and invasion activities involved in tumor cell motility. Curr. Protoc. Pharmacol. 41:12.11.1‐12.11.22. © 2008 by John Wiley & Sons, Inc.

Keywords: adhesion; migration; chemokines; integrins; selectins

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Steady‐State 96‐Well Plate Cell Adhesion Assay
  • Alternate Protocol 1: Steady‐State Cell Adhesion Assay on Slides: Drop Assay
  • Alternate Protocol 2: Cell Adhesion Under Flow Conditions
  • Basic Protocol 2: Transwell Migration Assay for Cells in Suspension
  • Alternate Protocol 3: Cell Migration Assay on Frame Filters for Adherent Cells
  • Alternate Protocol 4: Murine In Vivo Cell Migration Assay
  • Basic Protocol 3: Assessment of Compound Effects on In Vitro Tumor Cell Invasion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Steady‐State 96‐Well Plate Cell Adhesion Assay

  Materials
  • Substrates (available from BD Biosciences, Sigma‐Aldrich, R&D Systems, Calbiochem, Chemicon; store stocks solutions up to 1 year at –20°C), e.g.:
    • 150 mM fibronectin stock in PBS or other aqueous medium (e.g., tissue culture medium)
    • 5 mg/ml collagen stock in 20 mM acetic acid
    • 150 mM VCAM stock in PBS or other aqueous medium (e.g., tissue culture medium)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Chemokines (R&D Systems, Peprotech; resuspend to 10 µM in sterile PBS; store up to 6 months at –20°C in 5‐ to 10‐µl aliquots; avoid multiple freeze‐thaw cycles)
  • Test compound(s)
  • 2% (w/v) bovine serum albumin (BSA)
  • BCECF‐AM [2′,7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, acetoxymethyl ester] (Calbiochem, Molecular Probes)
  • Dimethylsulfoxide (DMSO)
  • Target cells of interest, e.g.:
    • Adherent cell lines (HEK‐293, 3T3 fibroblasts, MeWO, BLM)
    • Cells growing in suspension (Molt4, Jurkatt, Mono Mac‐1, THP‐1)
    • Primary cells (isolated human and mouse primary monocytes, B and T cells)
  • Depletion medium: serum‐free DMEM or RPMI‐1640 medium containing 0.5% (w/v) bovine serum albumin (BSA)
  • Tissue culture medium (DMEM or RPMI‐1640)
  • PBS (see recipe) containing 0.5% (w/v) sodium dodecyl sulfate (SDS)
  • 96‐well flat‐bottom plates with high protein‐capture capacity (e.g., Nunc, Costar, BD Falcon)
  • Orbital shaker (e.g., Belly Dancer, Stovall Life Science, http://www.slscience.com/)
  • Centrifuge with microtiter plate carrier
  • Fluorimeter (Millipore; Thermo Scientific; Tecan; BMG Labtech, http://www.bmglabtech.com)
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Alternate Protocol 1: Steady‐State Cell Adhesion Assay on Slides: Drop Assay

  • Fetal bovine serum (FBS)
  • Hanks] balanced salt solution (HBSS, e.g., Sigma, Invitrogen) containing 10 mM HEPES, pH 7.5
  • Hanks' balanced salt solution (HBSS, e.g., Sigma, Invitrogen) containing 10 mM HEPES, pH 7.5, and 1.5% (w/v) glutaraldehyde
  • 12‐well chamber slides (Nunc)
  • Humidified chamber: e.g., covered plastic container with moistened paper towels on the bottom
  • 37°C heating block
  • Image J software (download free at http://rsb.info.nih.gov/ij/)

Alternate Protocol 2: Cell Adhesion Under Flow Conditions

  • Substrate solution: 20 µg/ml fibronectin, 20 µg/ml collagen, or 4 µg/ml MadCAM (prepare from stock solutions described in protocol 1 materials list)
  • Tissue culture medium (DMEM or RPMI‐1640) containing 10% (v/v) FBS
  • Depletion medium: serum‐free DMEM or RPMI‐1640 containing 2% (w/v) BSA
  • Tissue culture medium (DMEM or RPMI‐1640) containing 50 µM EDTA
  • Tissue culture medium (DMEM or RPMI‐1640)
  • PBS (see recipe) containing 0.05% (w/v) sodium azide (prepare from 10% w/v NaN 3 stock in H 2O stored up to 1 year or longer protected from light in amber glass at room temperature)
  • 100‐mm petri dishes (e.g., Nunc, Falcon‐BD Systems, Sigma‐Aldrich)
  • Permanent marker
  • Parallel flow chamber (e.g., Glycotech, http://www.glycotech.com; see Fig. ) with syringe pump (e.g., Harvard Apparatus) and vacuum pump
  • Inverted light microscope
  • CCD camera (e.g., Cohu, http://www.cohu.com)

Basic Protocol 2: Transwell Migration Assay for Cells in Suspension

  Materials
  • Substrate (optional): endothelial cells (HUVEC or bEnd mouse brain endothelial cells), 5 µg/ml fibronectin, or 10 µg/ml collagen (prepare from stock solutions described in protocol 1 materials list)
  • Depletion medium: serum‐free DMEM or RPMI‐1640 containing 2% (w/v) BSA
  • Chemottractants, chemokines, or test compounds of interest (resuspend chemokines to 10 µM in sterile PBS and store frozen at –20°C for up to 6 months in 5‐ to 10‐µl aliquots to avoid repeated freeze‐thaw cycles)
  • Migration medium: DMEM or RPMI 1640 containing 25 mM HEPES and 0.1% (w/v) BSA
  • 0.5% (w/v) crystal violet in 20% (v/v) methanol (store at room temperature up to 6 months; use for adherent cells)
  • Transwells, 3‐, 5‐, or 8‐µm pore size (Costar, BD Biosystems, Nunc, Corning)
  • Centrifuge
  • Cotton swabs (for adherent cells)
  • Luminometer (Millipore; Thermo Scientific; Tecan; BMG Labtech, http://www.bmglabtech.com)
NOTE: Disposable 24‐well transmigration chambers are recommended for cells growing in suspension. The pore size depends on cell type (see Critical Parameters). Sterile conditions are usually unnecessary because cell migration experiments are conducted over a brief period of time (less than 4 hr). If assay must be adapted for adherent cells, see step 5, below; also see protocol 5.

Alternate Protocol 3: Cell Migration Assay on Frame Filters for Adherent Cells

  Materials
  • 20 µg/ml collagen VI or 5 µg/ml fibronectin (i.e., Sigma‐Aldrich, BD Biosciences), prepared in distilled H 2O
  • 5 mM tetrasodium EDTA
  • 0.05% (w/v) trypsin/5 mM tetrasodium EDTA
  • Migration medium: RPMI‐1640 or DMEM containing 25 mM HEPES and 0.1% (w/v) BSA
  • Chemoattractant or agonist test compounds in migration medium
  • 0.5% (w/v) crystal violet in 20% (v/v) methanol (store up to 6 months at room temperature)
  • Permanent marker
  • Frame filters, 8‐ or 10‐µm pore size (Neuro Probe Inc.)
  • Centrifuge
  • Surgical instruments, sutures, and wound clips or skin staples
  • Migration chamber (Neuro Probe Inc.)
  • Cell scraper
  • Densitometer

Alternate Protocol 4: Murine In Vivo Cell Migration Assay

  Materials
  • Cells of interest
  • Vital dye cell trackers (Molecular Probes, Calbiochem, Promega, Sigma‐Aldrich), e.g.:
    • BCECF‐AM (absorption/emission 475/535 nm)
    • CMTMR (541/565 nm)
    • CMFDA (492/517)
    • Calcein‐AM (494/517 nm)
  • Dimethylsulfoxide (DMSO), anhydrous
  • Phosphate‐buffered saline (PBS; see recipe)
  • Mice (C57BL/6, BALB/c; healthy animals of any age or gender)
  • Anesthetic (ketamine and xylazine; also see Donovan and Brown, )
  • Chemoattractant or agonist test compounds
  • Chemokine (optional; resuspend to 10 µM in sterile PBS; store up to 6 months at –20°C in small aliquots; avoid repeated freeze‐thaw cycles)
  • Tissue culture medium (DMEM or RPMI‐1640)
  • Erythrocyte lysis buffer: 0.85% (w/v) NH 4Cl in H 2O
  • Centrifuge
  • 100‐mm petri dish
  • 40‐µm nylon cell strainers
  • 15‐ml centrifuge tubes
  • Additional reagents and equipment for injection of mice (Donovan and Brown, ), anesthesia of mice (Donovan and Brown, ), euthanasia of mice (Donovan and Brown, ), survival surgery/splenectomy on mice (Reeves et al., ), and flow cytometry (Robinson et al., )

Basic Protocol 3: Assessment of Compound Effects on In Vitro Tumor Cell Invasion

  Materials
  • Matrigel (BD Biosciences)
  • Cells: e.g., tumor cells, neutrophils, eosinophils or endothelial cells
  • Chemoattractants or other test compounds
  • Depletion medium: serum‐free DMEM or RPMI‐1640 containing 0.5% (w/v) BSA
  • 4% (w/v) paraformaldehyde in PBS (see recipe for PBS)
  • 0.1% (w/v) crystal violet in 20% (v/v) methanol
  • 24‐well transmigration chambers (8‐µm pore size; e.g., Costar, BD Biosystems, Nunc, Corning)
  • Cotton swabs
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Figures

Videos

Literature Cited

   Donovan, J. and Brown, P. 1998. Anesthesia. Curr. Protoc. Immunol. 27: 1.4.1‐1.4.5.
   Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73: 1.6.1‐1.6.10.
   Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73: 1.8.1‐1.8.4.
   Frade, J.M., Mellado, M., del Real, G., Gutierrez‐Ramos, J.C., Lind, P., and Martinez, A.C. 1997. Characterization of the CCR2 chemokine receptor: Functional CCR2 receptor expression in B cells. J. Immunol. 159: 5576‐5584.
   Grabovsky, V., Feigelson, S., Chen, C., Bleijs, D.A. Peled, A., Cinamon, G., Baleux, F., Arenzana‐Seisdedos, F., Lapidot, T., van Kooyk, Y., Lobb, R.R., and Alon, R. 2000. Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J. Exp. Med. 192: 495‐506.
   Hernanz‐Falcon, P., Rodríguez‐Frade, J.M., Serrano, A., Juan, D., del Sol, A., Soriano, S.F., Roncal, F., Gomez, L., Valencia, A., Martinez, A.C., and Mellado, M. 2004. Identification of amino acid residues crucial for chemokine receptor dimerization. Nat. Immunol. 5: 216‐223.
   Ley, K., Laudanna, C., Cybulsky, M.I., and Nourhargh, S. 2007. Getting to the site of inflammation: The leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7: 678‐689.
   Mellado, M., Rodríguez‐Frade, J.M., Manes, S., and Martinez‐A., C. 2001. Chemokine signaling and functional responses: The role of receptor dimerization and TK pathway activation. Annu. Rev. Immunol. 19: 397‐421.
   Mellado, M., Martin de Ana, A., Gomez, L., Martinez‐A, C., and Rodríguez‐Frade, J.M. 2007. Chemokine receptor 2 blockade prevents asthma in a cynomolgus monkey model. J. Pharmacol. Exp. Ther. 324: 769‐775.
   Pachynski, R.K., Wu, S.W., Gunn, M.D., and Erle, D.J. 1998. Secondary lymphoid‐tissue chemokine (SLC) stimulates integrin alpha 4 beta 7‐mediated adhesion of lymphocytes to mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) under flow. J. Immunol. 161: 563‐571.
   Reeves, J.P., Reeves, P.A., Chin, L.T. 1991. Survival surgery: Removal of the spleen or thymus. Curr. Protoc. Immunol. 2: 1.10.1‐1.10.11.
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   Rodríguez‐Frade, J.M., del Real, G., Serrano, A., Hernanz‐Falcón, P., Soriano, S.F., Vila‐Coro, A.J., Martín de Ana, A:, Lucas, P., Prieto, I., Martínez‐A., C., and Mellado, M. 2004. Blocking HIV‐1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor EMBO J. 23: 66‐76.
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   Sanz‐Rodriguez, F., Hidalgo, A., and Teixido, J. 2001. Chemokine stromal cell‐derived factor‐1alpha modulates VLA‐4 integrin‐mediated multiple myeloma cell adhesion to CS‐1/fibronectin and VCAM‐1. Blood 97: 346‐351.
   Springer, T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76: 301‐314.
   Stein, J.V., Soriano, S.F., M'Rini, C., Nombela‐Arrieta, C., de Buitrago, G.G., Rodríguez‐Frade, J.M., Mellado, M., Girard, J.P., and Martinez‐A, C. 2003. CCR7‐mediated physiological lymphocyte homing involves activation of a tyrosine kinase pathway. Blood 101: 38‐44.
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Internet Resources
   http://www.ramicweb.com
  Web page of the Spanish Cell Adhesion and Migration Network (in English).
   http://bme.virginia.edu/ley/
  Page from the University of Virginia, with data on the molecules involved in the leukocyte adhesion cascade.
   http://apresslp.gvpi.net/apcyto/lpext.dll?f=templates&fn=main‐h.htm&2.0
  Cytokine Reference. An on‐line book with detailed information on cytokines, chemokines, growth factors, and other host defense mediators. Information organized by family, ligand, receptor, disease or cell type.
   http://www.neuro.wustl.edu/neuromuscular/lab/adhesion.htm#
  Detailed information on molecules involved in cell movement: selectins, integrins, immunoglobulin superfamily, and cadherins.
   http://www.cellmigration.org/index.shtml
  Cell Migration Gateway: a comprehensive and regularly updated resource for anyone interested in cell migration. Collaboration between the Cell Migration Consortium and Nature Publishing Group.
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