Fig. 6. (A) Measurements of unitary LFA-1–ICAM-1 unbinding forces. A series of AFM force measurements are shown. Traces 2 and 5 show molecular adhesion. Measurements of LFA-1–ICAM-1 unbinding forces were obtained under conditions that minimized contact between the 3A9 cell and the ICAM-1-coated surface. An adhesion frequency of less than 30% in the force measurements ensured that there is a >85% probability that the adhesion event is mediated by a single LFA-1–ICAM-1 complex (Tees et al., 2001). The specificity of the molecular interaction was confirmed by examining the frequency of adhesion in test and control experiments (Tees et al., 2001; Evans et al., 2001). Under identical experimental conditions, the addition of monoclonal antibodies against either LFA-1 or ICAM-1 significantly lowered the frequency of adhesion of both resting and activated cells. Moreover, both resting and stimulated 3A9 cells exhibited lower frequency of adhesion to immobilized bovine albumin than to immobilized ICAM-1. (B) Force histograms of unitary LFA-1–ICAM-1 unbinding forces of resting, PMA-stimulated and Mg²⁺/EGTA-treated 3A9 cells at low (73-98 pN/second) intermediate (1050-1500 pN/second) and high (21,000-35,000 pN/second) loading rates.