Biomembrane Force Probe to Quantitate Receptor-Ligand Interactions

The interaction between a T-cell receptor, TCR, and an antigenic peptide presented by the MHC, or major histocompatibility complex, on an antigen-presenting cell, APC, causes the T-cell to exert a mechanical force on the APC. This phenomenon is necessary for T-cell activation.

To quantify receptor-ligand interactions using a biomembrane force probe, BFP, begin with a slide-based custom cell chamber. Place the cell chamber under a microscope.

Inject suspensions of biotinylated swollen RBCs, probe glass beads coated with peptide-bound MHC-streptavidin-biotin complexes, and T-cells into the chamber. Position the probe, target, and helper buffer-filled micropipettes, around the chamber.

Aspirate a biotinylated RBC with the probe pipette, positioning it in a blank area. Aspirate a T-cell with the target micropipette, positioning it on the RBC's right. Aspirate a probe bead with the helper micropipette, and attach it to the RBC's apex. The bead's streptavidin binds to the RBC's biotin and forms a force probe.  

Align the T-cell with the force probe to allow the TCR to interact with its ligand ― the MHC-bound peptide ― and the T-cell to exert force on the RBC. Retract the T-cell to break the TCR-peptide bond, which deforms the RBC and displaces the bead.

Measure the force needed to break the bond ― a stronger bond needs a higher force and more time to break, indicating a longer lifetime.

To begin the BFP experiment, turn on the microscope and light source. Place the chamber onto the main microscope stage. The next step is to install all three micropipettes of the BFP. The probe to the left is to grab an RBC. The target to the right is to grab the cell, and the helper to the lower right serves to grab a bead.

To install, use a micro-injector to backfill a micropipette with experimental buffer. Take the micropipette off the pipette holder, and hold it at a lower place to allow liquid to drip from the tip. Quickly, insert the micropipette into the holder tip, making sure no air microbubble gets into the micropipette during the insertion.

Tighten the holder screw, then, mount each pipette holder onto its corresponding micromanipulator. Push the micropipette towards the chamber so that their tips enter the chamber buffer area. Adjust the position of the micropipette, and find them under the microscope field of view.

Move around the chamber holder's stage to find the colonies of the three elements one-by-one. Adjust the position of the corresponding micropipette by turning the knobs of the manipulators to let the tip of the micropipette approach one cell or bead. Aspirate the cell or bead by adjusting the pressure inside the corresponding micropipette. All three micropipettes will capture their corresponding elements.

Move around the chamber holder stage to find an open space away from the colonies of the injected elements where the experiment will be performed.

Switch the microscope visual mode to visualize the image on the computer program. Move all three elements on the pipette tips into the program's vision field. Next, align the probe bead and the RBC and carefully maneuver the probe bead to the apex of the RBC. Now, briefly impinge the bead onto the RBC and gently retract.

Adjust the pressure of the helper micropipette to gently blow the bead away so that it would be left glued onto the RBC apex. Move the helper micropipette away, and align the target, and probe bead.

On the program, in the Vision Field window, use the program tools to measure the respective radii of the probe micropipette, the RBC, and the circular contact area between the RBC and the probe bead. These values allow for the estimation of the spring constant of the RBC by the equation listed in the text protocol.

After entering the desired RBC spring constant into the program, draw a horizontal line across the RBC apex which will yield a curve in the adjacent window indicating the brightness of each pixel along this line. Drag the threshold line to be at around half the depth of the curve.

Next, select the desired experiment mode. Set the parameters as desired. Click Start which allows the program to move the target pipette and drive the target in and out of contact with the probe.

Data collection will be performed in parallel which records the position of the probe bead in real-time. Stop the program by clicking on the button Stop Experiment at which time a window will pop out to allow saving the acquired data.