Successive detection of insulin-like growth factor-II bound to receptors on a living cell surface using an AFM

In this study, we have developed a method of mechanical force detection for ligands bound to receptors on a cell surface, both of which are involved in a signal transduction pathway. This pathway is an autocrine pathway, involving the production of insulin-like growth factor-II (IGF-II) and activation of the IGF-I receptor, involved in myoblast differentiation induced by MyoD in C3H10T1/2 mouse mesenchymal stem cells. Differentiation of C3H10T1/2 was induced with the DNA demethylation agent 5-azacytidine (5-aza). The etched AFM tip used in the force detection had a flat surface of which about 10 µm(2) was in contact with a cell surface. The forces required to rupture the interactions of IGF-IIs on a cell and anti mouse IGF-II polyclonal antibody immobilized on an etched AFM tip were measured within 5 days of induction of differentiation. The mean unbinding force for a single paired antibody-ligand on a cell was about 81 pN, which was measured at a force loading rate of about 440 nN/s. The percentage of unbinding forces over 100 pN increased to 32% after 2 days from the addition of 5-aza to the medium. This method could be used in non-invasive and successive evaluation of a living cell's behavior.