FIGURE 3:
Experimental setup for single-molecule force microscopy of αIIbβ3–RGD interaction. (A) Schematic and bottom view microgragh of AFM microcantilever functionalized with αIIbβ3 integrin above RGDP2Y2R binding sites on cell membranes. For the 1321N1 astrocytes, experiments were performed in DMEM containing 5% FBS with addition of 10 mM HEPES (pH 7.4). For CHO-K1, we used Tyrode’s buffer (Sigma-Aldrich) containing 1 mM CaCl2, 1 mM MgCl2, 0.1% glucose, and 0.1% BSA. For Mn2+ activation, 1 mM CaCl2 and 1 mM MgCl2 were replaced with 2 mM MnCl2. (B) A typical cantilever-RGDP2Y2R 1321N1 force scan. Gray and black traces are engagement and retract traces, receptively. Fu is the unbinding force on the retract trace. The lower panel shows the four stages of stretching and rupturing a single ligand-receptor complex using the AFM: 1) AFM-cell surface engagement, 2) retraction, 3) extension, and 4) rupture.