Cell‐Substrate Adhesion Assays

Martin J. Humphries1

1 University of Manchester, Manchester
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 9.1
DOI:  10.1002/0471143030.cb0901s00
Online Posting Date:  May, 2001
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Abstract

In the intact organism, cells adhere to a variety of substrates. It is possible to investigate the nature of the molecules to which cells adhere in two different assays. In the first protocol presented in this unit, physical adhesion of a cell is assessed by determining the extent to which the cell spreads on a defined substrate‐‐the plate is coated with the test substance, cells are added and allowed time to attach and spread, and the extent of spreading is assessed using phase contrast microscopy. In a second assay, an aliquot of cells is added to the well of a microtiter plate coated with a test adhesion molecule and the cells are allowed to attach. Nonattached cells are removed, and the percent of added cells attached to the substrate is quantified by crystal violet staining.

     
 
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Table of Contents

  • Basic Protocol 1: Spreading Assay
  • Basic Protocol 2: Attachment Assay
  • Support Protocol 1: Preparation of Peptide‐Protein Conjugates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Spreading Assay

  Materials
  • Dulbecco's PBS (DPBS; Life Technologies; appendix 2A)
  • 10 mg/ml heat‐denatured BSA solution (see recipe)
  • Cells of interest
  • Dulbecco's modified Eagle medium with 25 mM HEPES (DMEM/HEPES; Life Technologies; appendix 2B), prewarmed to 37°C and gassed with 5% to 10% (v/v) CO 2
  • 2× exogenous agent (e.g., antibody, peptides) in DPBS
  • 5% (w/v) glutaraldehyde (dilute 50% stock solution with water)
  • CMF‐DPBS/NaN 3: divalent cation–free Dulbecco's PBS (CMF‐DPBS; Life Technologies; appendix 2A) containing 0.05% (w/v) sodium azide (NaN 3)
  • 96‐well tissue culture microtiter plates (Costar)
  • Aspirator
  • Multichannel pipettor
  • 15‐ml polypropylene tubes
  • Glass coverslips
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.1)

Basic Protocol 2: Attachment Assay

  Materials
  • Dulbecco's PBS (DPBS; Life Technologies; appendix 2A)
  • 10 mg/ml heat‐denatured BSA solution (see recipe)
  • Cells of interest
  • Dulbecco's modified Eagle medium with 25 mM HEPES (DMEM/HEPES; Life Technologies; appendix 2B), prewarmed to 37°C and gassed with 5% to 10% (v/v) CO 2
  • 2× exogenous agent (e.g., antibody, peptides) in DPBS
  • 5% (w/v) glutaraldehyde (dilute 50% stock solution with water)
  • 0.1% (w/v) crystal violet solution (see recipe)
  • 10% (v/v) acetic acid
  • 96‐well tissue culture microtiter plates (Costar)
  • Aspirator
  • 15‐ml polypropylene tubes
  • Microtiter plate reader
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.1)

Support Protocol 1: Preparation of Peptide‐Protein Conjugates

  Materials
  • 3 mg/ml N‐succinimidyl 3‐(2‐pyridyldithio)propionate (SPDP; Pierce) in ethanol
  • Divalent cation–free Dulbecco's PBS (CMF‐DPBS; Life Technologies; appendix 2A)
  • Cysteine‐containing peptide, solid
  • Dulbecco's PBS (DPBS; Life Technologies; appendix 2A)
  • 10‐ml Sephadex G25 columns (PD‐10, Amersham Pharmacia Biotech)
  • Additional reagents and equipment for dialysis ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Chan, B.M.C., Elices, M.J., Murphy, E., and Hemler, M.E. 1992. Adhesion to vascular cell adhesion molecule 1 and fibronectin. Comparison of α4β1 (VLA‐4) and α4β7 on the human B cell line JY. J. Biol. Chem. 267:8366‐8370.
   Dustin, M.L. and Springer, T.A. 1989. T cell receptor cross‐linking transiently stimulates adhesiveness through LFA‐1. Nature 341:619‐624.
   Humphries, M.J., Mould, A.P., and Weston, S.A. 1994. Conjugation of synthetic peptides to carrier proteins for cell adhesion studies. J. Tissue Culture Methods. 16:239‐242.
   Kueng, W., Silber, E., and Eppenberger, U. 1989. Quantification of cells cultured on 96‐well plates. Anal. Biochem. 182:16‐19.
   Prater, C.A., Plotkin, J., Jaye, D., and Frazier, W.A. 1991. The properdin‐like type I repeats of human thrombospondin contain a cell attachment site. J. Cell Biol. 112:1031‐1040.
   Yamada, K.M. and Kennedy, D.W. 1984. Dualistic nature of adhesive protein function: Fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function. J. Cell Biol. 99:29‐36.
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