Measurement of Cellular Adhesion Under Static Conditions

James L. Mobley1, Yoji Shimizu1

1 University of Minnesota Medical School, Minneapolis, Minnesota
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.28
DOI:  10.1002/0471142735.im0728s37
Online Posting Date:  May, 2001
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Abstract

This unit describes procedures for measuring the adhesion of lymphoid cells to immobilized ligands or adherent cell monolayers. The basic protocol, which is optimized for human T cells, can be easily modified to study almost any cell type in suspension if the corresponding adhesive ligand is available. Alternate protocols describe modifications to the basic protocol that allow analysis of cell adhesion in the presence of stimulators of lymphocyte activation or monoclonal antibodies (MAbs) that react with adhesion molecules or their ligands, or analysis of cell adhesion to adherent cell monolayers. A support protocol is provided for determining the optimal concentration of adhesion ligands and choosing the appropriate type of microtiter plate for static adhesion assays.

     
 
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Table of Contents

  • Basic Protocol 1: Measuring Cell Adhesion to Immobilized Ligands
  • Alternate Protocol 1: Analyzing Cell Adhesion in the Presence of Stimulators of Lymphocyte Activation
  • Alternate Protocol 2: Analyzing Cell Adhesion in the Presence of Stimulatory or Inhibitory MAbs
  • Alternate Protocol 3: Analyzing Cell Adhesion to Adherent Cell Monolayers
  • Support Protocol 1: Optimizing Adhesion Ligand Concentrations and Type of Microtiter Plate
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measuring Cell Adhesion to Immobilized Ligands

  MaterialsFor suppliers, see appendix 55
  • Purified ligand (e.g., fibronectin or ICAM‐1; Table 7.28.2)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 900 µM Ca2+ and 500 µM Mg2+ (store at room temperature)
  • PBS/HSA: PBS/Ca2+/Mg2+ with 0.5% (v/v) human serum albumin (store at 4°C)
  • PBS/BSA: PBS/Ca2+/Mg2+ with 2.5% (w/v) bovine serum albumin (Calbiochem; store at 4°C)
  • Purified human T cells (unit 7.4)
  • Complete RPMI medium ( appendix 2A)
  • 1 µCi/µl Na 251CrO 4 in normal saline (∼500 µCi/µg, DuPont NEN or Amersham)
  • 1% (v/v) Triton X‐100 (Sigma) in H 2O
  • Multichannel pipettor and disposable tips
  • 96‐well flat‐bottom microtiter plate (Costar or Nunc MaxiSorp F96)
  • Syringe pipet (Micromatic, Popper and Sons)
  • Manifold, 8‐tip bent (Micromatic, Popper and Sons)
  • Manifold, 8‐tip straight (Drummond Scientific)
  • 15‐ml polystyrene centrifuge tubes (Falcon)
  • IEC Centra‐7R refrigerated centrifuge with #216 rotor and 15‐ml‐tube adaptors (or equivalent)
  • Repeating pipet (e.g., Oxford Labware)
  • Inverted light microscope
  • Supernatant collection system (Skatron; optional)
  • γ counter (Beckman GAM5500 or equivalent)
  • Additional reagents and equipment for counting cells ( appendix 3B)
    Table 7.8.2   MaterialsSelected Commercial Sources of Purified Adhesion Ligands

    Ligand Human Mouse
    Collagen type IV GIBCO/BRL GIBCO/BRL
    Fibrinogen Sigma (human type I) Sigma (mouse plasma)
    Fibronectin GIBCO/BRL GIBCO/BRL
    Hyaluronic acid Sigma (human umbilical) Sigma (bovine tracheal)
    Laminin GIBCO/BRL GIBCO/BRL
    Thrombospondin GIBCO/BRL
    Vitronectin GIBCO/BRL GIBCO/BRL

Alternate Protocol 1: Analyzing Cell Adhesion in the Presence of Stimulators of Lymphocyte Activation

  Additional MaterialsFor common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Fibronectin (FN)
  • Murine MAb specific for human CD3 (e.g., OKT3, ATCC #CRL 8001), without azide or other preservatives
  • Goat anti‐mouse Ig (GAMIg, Cappel), without azide or other preservatives
  • 100 µg/ml phorbol 12‐myristate 13‐acetate (PMA; Calbiochem) in DMSO (store at −70°C; dilute in PBS/HSA immediately before use)
NOTE: Because many adhesive interactions depend on proper metabolic activity, preservatives that inhibit metabolism may interfere with these adhesion assays; therefore, antibodies should not be stored in sodium azide or other preservatives.

Alternate Protocol 2: Analyzing Cell Adhesion in the Presence of Stimulatory or Inhibitory MAbs

  Additional Materials
  • Control MAbs, without azide or other preservatives
  • Test MAbs, without azide or other preservatives
NOTE: Because many adhesive interactions depend on proper metabolic activity, preservatives that inhibit metabolism may interfere with these adhesion assays; therefore, antibodies should not be stored in sodium azide or other preservatives.

Alternate Protocol 3: Analyzing Cell Adhesion to Adherent Cell Monolayers

  Additional MaterialsFor suppliers, see appendix 55.
  • Adherent cells (e.g., HUVEC, unit 6.12), suspended in appropriate tissue culture medium
  • 24‐well tissue culture plates (Costar)
  • 100 µg/ml phorbol 12‐myristate 13‐acetate (PMA; Calbiochem) in DMSO (store at −70°C; dilute in PBS/HSA immediately before use)

Support Protocol 1: Optimizing Adhesion Ligand Concentrations and Type of Microtiter Plate

  Additional Materials
    For suppliers, see appendix 55.
  • 12 × 75–mm test tubes (Falcon)
  • 96‐well tissue culture–treated microtiter plate (Costar)
  • 96‐well ELISA‐treated microtiter plate (Nunc)
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Figures

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Literature Cited

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Key References
   Shimizu et al., 1990. See above.
  Details the adhesion assay outlined here with multiple variations.
   Springer, 1990. See above.
  Comprehensive review of immunologically relevant adhesion molecules.
   Springer, T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76:301‐314.
  Reviews traffic signals controlling emigratory behavior of leukocytes and the sequential signals associated with lymphocyte recirculation.
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