Force Spectroscopy: Coupling a Single Live Cell onto the Cantilever

This section describes a procedure for attaching a single live cell onto the AFM cantilever (Fig. 5). Cell-functionalized cantilevers are used to study cell-cell interaction as well as cell-substrate interaction. Our approach was based on earlier work by Benoit et al. (2000) and took advantage of the fact that many cells adhere well to the Con A-functionalized cantilever. The experimental protocol outlined below uses a 3A9 cell-functionalized cantilever to probe a substrate that has been coated with intercellular adhesion molecule-1 (ICAM-1).

MATERIALS

Biotinylated concanavalin A (Con A) (Sigma), recombinant mouse ICAM-1 (R& D Systems), 35 mm plastic petri dishes, mouse lymphoma cell line 3A9, RPMI 1640 medium (supplemented with 10% FCS and 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)), sodium bicarbonate buffer (0.1 M, pH 8.3).

METHODS

• Prepare ICAM-1-coated dishes for 3A9 cell adhesion studies.

• Place a 30 µl drop of ICAM-1 (0.05 mg/ml in sodium bicarbonate, pH 8.3) on the center of a 35 mm plastic petri dish.

• Incubate overnight at 37°C in a humidified incubator.

• Mark the ICAM-1 coated area and rinse the dish three times in PBS.

• Add 2 ml of the RPMI medium to the ICAM-1 coated dish for 30 minutes prior to the experiment. The fetal calf serum in the medium will block non-specific binding to the exposed surface of the dish.

• Functionalize the AFM tip with biotinylated-Con A prior to the measurement (see Protocol 1).

• Perform cantilever calibration outside the ICAM-1 coated area in 2 ml RPMI medium (see Section II). Inject 50 mL of 3A9 cells (10 5 cells/mL) into the petri dish.

• The procedure for attaching the 3A9 cell is done with the aid of an inverted light microscope. To attach the cell to the cantilever, align the tip of cantilever over the cell and lower the Con A-functionalized cantilever onto the cell for approximately one second. This should be sufficient time for the cell to adhere to the cantilever.

• Move the 3A9 cell-functionalized cantilever over to the ICAM-1 coated area to carry out adhesion studies as described Protocol 2.

Note:

• Cantilever calibration should be done before cell coupling;

• Coupling the cell outside the ICAM-1-coated area will facilitate its attachment to the cantilever;

• Make sure that the cell is behind the AFM tip as lowering the sharp tip onto the cell surface may damage the cell (Fig. 5B);

Using this protocol, we were able to measure the unbinding force of individual integrin LFA-1/ICAM-1 (Zhang et al., 2002; Wojcikiewicz et al., 2003) and integrin a5 b1/fibronectin (Li et al., 2003) bonds.