Force Spectroscopy: AFM Measurements on Living Cells

Because the properties of purified molecules may not be the same as when they are under physiological conditions, it is best to study ligand-receptor interactions on living cells. This section describes the use of a functionalized cantilever to probe the surface of a live cell. Using AFM, it is possible to acquire direct measurements of adhesion down to the level of single molecule pairs, unlike earlier cell adhesion assay studies and biochemical methods that relied on indirect measurements and could only access receptor group dynamics. Lehenkari and Horton (1999) were able to measure RGD (arginine-glycine-aspartic acid)-integrin binding on the surface of osteoblasts and osteoclasts, and recently, we measured the binding force between concanavalin A and its receptor on the surface of NIH-3T3 fibroblasts (Chen and Moy, 2000). A brief generalizable protocol used to perform this study is as follows.

MATERIALS

Biotinylated concanavalin A (con A) (Sigma), NIH-3T3 fibroblast cells plated in plastic tissue culture dish, glucose-free RPMI 1640 medium (supplemented with 0.01% BSA and 0.01 mM MnCl 2).

METHODS

• Functionalize the AFM tip with streptavidin (see Protocol 1).

• Incubate streptavidin-functionalized cantilever in biotinylated-Con A (0.5 mg/ml in PBS) for 10 minutes at room temperature.

• Lower Con A-functionalized cantilever onto the surface of NIH-3T3 cell to initiate the binding of Con A to its receptors on the cell surface.

• To prevent potential competitive binding of glucose with the Con A-functionalized tip and Con A receptors on the cell, the AFM measurements are performed in glucose-free RPMI media supplemented with 0.01% BSA and 0.01 mM MnCl 2.

Compared to measurements on agarose beads, cells have much longer regions of stretch before final separation between the tip and membrane (compare Fig. 4 with Fig. 3). This observation suggests that the Con A receptors are anchored to cell membrane via tethers that stretch as the receptors are pulled.

6-Coupling a Single Live Cell onto the Cantilever