Atomic force microscopy measurements of protein-ligand interactions on living cells.
Cell adhesion receptors are expressed on the surface of cells and can mediate binding to other cells and to the extracellular matrix. Here, we describe in detail the use of atomic force microscopy (AFM)-based force spectroscopy for studying cell detachment forces on living leukocytes. With this technique it is now possible to measure force with resolution down to the level of individual molecules. AFM force spectroscopy is particularly well suited for research in cell adhesion, which has relevance in both the medical and life sciences including immunology, cancer and stem cell research, and human pharmacology. Along with its limitations, we herein, describe how the rupture force of a single complex formed between the integrin receptor leukocyte function-associated antigen (LFA)-1, expressed on the surface of a living leukocyte, and immobilized intercellular adhesion molecule-1 (ICAM-1) was measured. With only minor modifications this protocol can be used to study other adhesion receptors on almost any mammalian cell or bacterial system. This protocol is also suitable for studying single-molecule de-adhesion events in cell-free systems as well as between two living cells.
Eibl
Robert H
RH
Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL, USA.
Moy
Vincent T
VT
D002448:Cell Adhesion; D018914:Cells, Immobilized; D003627:Data Interpretation, Statistical; D006801:Humans; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007962:Leukocytes / Q000378:metabolism; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*
D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D011506:Proteins; D018799:Intercellular Adhesion Molecule-1