Cell Adhesion Under Hydrodynamic Flow Conditions

Priya K. Gopalan1, David A. Jones1, Larry V. McIntire2, C. Wayne Smith3

1 Rice University, Baylor College of Medicine, Houston, Texas, 2 Rice University, Houston, Texas, 3 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.29
DOI:  10.1002/0471142735.im0729s15
Online Posting Date:  May, 2001
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Abstract

This unit describes a hydrodynamic assay to study the relative importance of various receptor/ligand interactions in cell‐cell and cell‐substrate adhesion and to quantitate the strength of their binding. The basic protocol describes how to assemble the single‐chamber flow system with the substrate, add the cells in suspension, and record the experiment on videotape. Alternate protocols present assays to determine how monoclonal antibodies and stimulating and inhibiting agents affect the substrate and the perfusing cells in suspension. Another alternate protocol details the use of the double‐chamber flow system. Support protocols describe how to construct the single‐ and double‐chamber flow systems and how to analyze the data from an experiment. Recording and analyzing the flow experiment requires the use of video equipment and, optionally, a computer and imaging software.

     
 
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Table of Contents

  • Basic Protocol 1: Parallel Plate Flow Assay to Measure Cell Adhesion
  • Alternate Protocol 1: Measurement of the Effects of Substrate‐Specific Monoclonal Antibodies or Activating/Inhibiting Substances on Cell Adhesion
  • Alternate Protocol 2: Measurement of the Effects of Perfusion Cell–Specific Monoclonal Antibodies or Activating/Inhibiting Substances on Cell Adhesion
  • Alternate Protocol 3: Double Flow Assay to Measure Cell Adhesion
  • Support Protocol 1: Construction of a Chamber with a Single Flow Section
  • Support Protocol 2: Construction of a Chamber with Two Flow Sections
  • Support Protocol 3: Data Analysis
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Parallel Plate Flow Assay to Measure Cell Adhesion

  Materials
  • 70% ethanol
  • Perfusion buffer containing Ca2+: e.g., PBS with Ca2+ ( appendix 2A), RPMI 1640, or M199
  • Bleach
  • Isolated cells in perfusion buffer at a concentration of 107 cells/ml: e.g., human neutrophils (unit 7.23), or human T cells (unit 7.4)
  • 35‐mm tissue culture dishes coated with substrate (e.g., immobilized purified ligand, units 3.5 & 7.3, Table 97.80.4711, and Table 97.80.4711, or cell monolayer such as human umbilical vein endothelial cells [HUVEC]—e.g., ECV304, ATCC)
  • Polycarbonate base with single flow section and tubing (see protocol 5 and Fig. and Fig. A)
  • Vacuum grease
  • Gasket for single flow section (see protocol 5 )
  • Pen or pencil
  • Lens paper or Kimwipes
  • Plastic stopcocks: 2 three‐way and 2 or 3 one‐way
  • 16‐G needle
  • 35‐mm tissue culture dishes, sterile
  • Two‐holed rubber stopper (e.g., Baxter Healthcare, size 0 or size to fit Erlenmeyer flask)
  • 0.125‐in.‐o.d. × 2 in.‐long tubing of any material
  • 25‐ml Erlenmeyer flask
  • Syringe pump (e.g., Harvard Apparatus), and glass syringe (larger than the feeder syringe) with Luer‐Lok tip
  • Disposable transfer pipets, sterile
  • Video microscopy system consisting of:
  • Phase‐contrast inverted microscope with 20× and 40× objectives and a video camera
  • Incubator or stage heater (optional)
  • Monitor
  • Video timer/titler (or any other timer)
  • Video cassette recorder
  • Videotape
  • Disposable Luer‐Lok syringes (feeder syringe; minimum size calculated in step ), sterile
  • Two large (e.g., 35‐ml) disposable Luer‐Lok syringes, sterile
  • 100‐ml beaker
  • Ring stand or masking tape

Alternate Protocol 1: Measurement of the Effects of Substrate‐Specific Monoclonal Antibodies or Activating/Inhibiting Substances on Cell Adhesion

  • Monoclonal antibodies: experimental and isotype‐matched Ig control (e.g., see Table 97.80.4711)
  • Activating or inhibiting agent (e.g., see Table 97.80.4711)

Alternate Protocol 2: Measurement of the Effects of Perfusion Cell–Specific Monoclonal Antibodies or Activating/Inhibiting Substances on Cell Adhesion

  • Monoclonal antibodies: experimental and isotype‐matched Ig control (e.g., see Table 97.80.4711)
  • Activating or inhibiting agent (e.g., see Table 97.80.4711)
  • Additional reagents and equipment for flow cytometry (unit 5.4)

Alternate Protocol 3: Double Flow Assay to Measure Cell Adhesion

  • Polycarbonate base with two separate flow sections and tubing (see protocol 6, Fig. , Fig. , and Fig. B)
  • Gasket cut for two separate flow sections (see protocol 6 and Fig. )
  • 2 three‐way plastic stopcocks
  • Two‐way plastic stopcock (optional)
  • Two‐holed rubber stopper (e.g., Baxter Healthcare size 0 or size to fit Erlenmeyer flask)
  • 0.125‐in.‐o.d. × 2‐in.‐long tubing of any material
  • 25‐ml Erlenmeyer flask
  • Dual syringe pump (e.g., Harvard Apparatus) and glass syringes (larger than the feeder syringe) with Luer‐Lok tips
  • Disposable Luer‐Lok syringes, large and small, sterile

Support Protocol 1: Construction of a Chamber with a Single Flow Section

  Materials
  • Piece of polycarbonate or other transparent plastic at least 1 3/8 in. × 1 3/8 in. × 1 1/8 in.
  • 0.005‐in.‐thick sheet of medical‐grade silicone rubber (e.g., PharmElast, S.F. Medical)
  • Ballpoint pen
  • Scissors
  • Scalpel
  • 16‐G needle with the bevel cut off
  • 4 Pharmaseal extension tubes (Baxter Healthcare)
  • 1/16‐in.‐i.d. × 10‐in.‐long gas‐impermeable (e.g., silicone) tubing

Support Protocol 2: Construction of a Chamber with Two Flow Sections

  Materials
  • Piece of polycarbonate or other transparent plastic at least 1 5/16 in. × 1 5/16 in. × ½ in.
  • 0.005‐in.‐thick sheet of medical‐grade silicone rubber (e.g., PharmElast, S.F. Medical)
  • Ballpoint pen
  • Scissors
  • Scalpel
  • 16‐G needle with the bevel cut off
  • 3 Pharmaseal extension tubes (Baxter Healthcare)
  • Two 1/16‐in.‐i.d. × 10‐in.‐long pieces of gas‐impermeable (e.g., silicone) tubing,
  • 5 plastic 90° threaded fittings (e.g., mounting Luer bulkhead 90° polypropylene fittings, Value Plastic)

Support Protocol 3: Data Analysis

  • Glass slide with micrometer scale
  • Computer and image analysis software (e.g., Bioscan OPTIMAS, Inovision CellTrack, NIH Image; optional)
  • Ruler (optional)
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Figures

Videos

Literature Cited

Literature Cited
   Abbassi, O., Lane, C.L., Krater, S., Kishimoto, T.K., Anderson, D.C., McIntire, L.V., and Smith, C.W. 1991. Canine neutrophil margination mediated by lectin adhesion molecule‐1 in vitro. J. Immunol. 147:2107‐2115.
   Abbassi, O., Kishimoto, T.K., McIntire, L.V., Anderson, D.C., and Smith, C.W. 1993. E‐selectin supports neutrophil rolling in vitro under conditions of flow. J. Clin. Immunol. 92:2719‐2730.
   Alevriadou, B.R., Moake, J.L., Turner, N.A., Ruggeri, Z.M., Folie, B.J., Phillips, M.D., Schreiber, A.B., Hrinda, M.E., and McIntire, L.V. 1993. Real‐time analysis of shear‐dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets. Blood 81:1263‐1276.
   Anderson, D.C., Abbassi, O., Kishimoto, T.K., Koenig, J.M., McIntire, L.V., and Smith, C.W. 1991. Diminished lectin‐, epidermal growth factor‐, complement binding domain‐cell adhesion molecule‐1 on neonatal neutrophils underlies their impaired CD18‐independent adhesion to endothelial cells in vitro. J. Immunol. 146:3372‐3379.
   Buttrum, S.M., Hatton, R., and Nash, G.B. 1993. Selectin‐mediated rolling of neutrophils on immobilized platelets. Blood 82:1165‐1174.
   Cozens‐Roberts, C., Quinn, J.A., and Lauffenburger, D.A. 1990. Receptor‐mediated adhesion phenomena: Model studies with the radial‐flow detachment assay. Biophys. J. 58:107‐125.
   Dewey, C.F. Jr., Bussolari, S.R., Gimbrone, M.A. Jr., and Davies, P.F. 1981. The dynamic response of vascular endothelial cells to fluid shear stress. J. Biomech. Eng. 103:177‐185.
   Jones, D.A., Abbassi, O., McIntire, L.V., McEver, R.P., and Smith, C.W. 1993. P‐selectin mediates neutrophil rolling on histamine‐stimulated endothelial cells. Biophys. J. 65:1560‐1569.
   Jones, D.A., McIntire, L.V., Smith, C.W., and Picker, L.J. 1994a. A two‐step adhesion cascade for T‐cell/endothelial cell interactions under flow conditions. J. Clin. Invest. 94:2443‐2450.
   Jones, D.A., Smith, C.W., and McIntire, L.V. 1994b. Flow modulation of receptor function and leukocyte adhesion to endothelial cells. In Cell Mechanics and Cellular Engineering (V. Mow and R. Hochmuth eds.) pp. 70‐94. Springer‐Verlag, New York.
   Kaplanski, G., Farnarier, C., Tissot, O., Pierres, A., Benoliel, A., Alessi, M., Kaplanski, S., and Bongrand, P. 1993. Granulocyte‐endothelium initial adhesion. Biophys. J. 64:1922‐1933.
   Lawrence, M.B. and Springer, T.A. 1991. Leukocytes roll on a selectin at physiologic flow rates: Distinction from and prerequisite for adhesion through integrins. Cell 65:859‐873.
   Lawrence, M.B. and Springer, T.A. 1993. Neutrophils roll on E‐selectin. J. Immunol. 151:6338‐6346.
   Lawrence, M.B., McIntire, L.V., and Eskin, S.G. 1987. Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion. Blood 70:1284‐1290.
   Luscinskas, F.W., Kansas, G.S., Ding, H., Pizcueta, P., Schleiffenbaum, B.E., Tedder, T.F., and Gimbrone, M.A. Jr. 1994. Monocyte rolling, arrest and spreading on IL‐4‐activated vascular endothelium under flow is mediated via sequential action of L‐selectin, β1‐integrins and β2‐integrins. J. Cell Biol. 125:1417‐1427.
   Smith, C.W., Kishimoto, T.K., Abbassi, O., Hughes, B., Rothlein, R., McIntire, L.V., and Butcher, E., Anderson, D.C. 1991. Chemotactic factors regulate lectin adhesion molecule 1 (LECAM‐1)‐dependent neutrophil adhesion to cytokine‐stimulated endothelial cells in vitro. J. Clin. Invest. 87:609‐618.
   Truskey, G.A. and Pirone, J.S. 1990. The effect of fluid shear stress upon cell adhesion to fibronectin‐treated surfaces. J. Biomed. Matl. Res. 24:1333‐1353.
   Wechezak, A.R., Wright, T.N., Viggers, R.F., and Sauvage, L.R. 1989. Endothelial adherence under shear stress is dependent upon microfilament reorganization. J. Cell. Physiol. 139:136‐146.
Key References
   Jones et al., 1994. See above.
  Reviews leukocyte adhesion molecules and their ligands, and evaluates different assays used to study leukocyte adhesion.
   Lawrence et al., 1987. See above.
  Describes the parallel plate flow chamber, including the parameters used and the theory behind it.
   Springer, T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76:301‐314.
  Reviews the major adhesion molecules of the immune system, many of which can be studied with the hydrodynamic adhesion assay.
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