Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

This protocol describes how best to functionalize the AFM cantilevers using single T cells and solid particles. These tips can then be used to probe single-pair dendritic cell-T cell interactions and to monitor cellular responses of fickle size, respectively. A main advantage of this technique is that it uses a biocompatible glue for single T cell functionalization of cantilevers.

This glue is inert to eukaryotic cells, thus keeping T cells in their baseline activation stage. These methods provide insight into immune cell activation and complex intercellular events, such as immune synapse formation. The methods can also be extended to other systems involving cell-cell or cell-surface interactions.

For someone that is new to this technique, it is important to make sure that the T cells are in good condition prior to use and that they are pretreated with IL-2 overnight. Additionally, when using the biocompatible glue, move quickly to protect it from unnecessary oxidation. To begin this procedure, prepare the single T cells for microscopy by following along in the text protocol.

Then, prepare glass coverslips seeded with DC2.4 cells, and incubate the cells overnight in a humidified chamber at 37 degrees Celsius and 5%CO2. Clean soft, tipless cantilevers with a low spring constant using piranha treatment, plasma cleaning, or UV-ozone cleaning. Then, mount the cleaned cantilever to the AFM scanning head.

Next, prepare a clean sample chamber, and fill it with pure water. Calibrate the cantilever in the water solution by first running a force curve on the glass substrate to obtain the sensitivity of the cantilever. Then, record a thermal noise spectrum to extract the spring constant.

When the cantilever is properly calibrated, remove the AFM scanning head from the solution. Wash the mounted cantilever with a few drops of pure ethanol, and keep the cantilever dry on the scanning head. Preheat a living cell environment enclosure to 37 degrees Celsius with 5%CO2.

Then, mount the glass coverslip seeded with DC2.4 cells to the sample chamber assembly, and immediately add 600 microliters of Medium B to the chamber. Place the completed assembly onto the AFM sample stage. Add human IL-2-incubated CD4-positive T cells into the sample chamber.

Wait until the targeting T cells are fully settled on the bottom of the coverslip, and then bring the cells into the field of view under the microscope. Now, add a two-microliter drop of biocompatible glue onto the end of the mounted cantilever with a pipette. Once the biocompatible glue has been applied to the cantilever, the subsequent steps need to be finished as soon as possible in order to maximize adhesion.

Quickly place the scanning head on the sample stage, immersing the glue-coated cantilever in the solution. Move the sample stage until a health T cell is located beneath the tip of the cantilever. Then, finely adjust its position by moving the scanning head.

Next, lower the cantilever manually. Begin with a 50-micron step size, and then decrease to 10, five, and two and finally 0.5 microns gradually, as indicated by the sharpness of the cantilever image. Now, hold the position of the stepper motors, and adjust the positioning of the scanning head to better align the cantilever tip and the cell.

Continue until firm contact is indicated by a small displacement of the laser's position, corresponding to a typical force of 0.5 to 1.5 nanonewtons. After 30 seconds of contact, retract the cantilever. If the cell moves with the cantilever, the attachment was successful.

If not, repeat the adhesion step up to three times before switching to a new cantilever. Position the attached T cell above a DC2.4 cell by moving the sample stage and the scanning head. After setting up proper parameters, run force spectroscopy over the sample.

For a new round of probing, mount a new, clean cantilever. Calibrate it in pure water, and then go back to a T cell-dendritic cell pair and repeat the scan. Mount a cleaned glass coverslip to the sample chamber assembly.

To the left side of the coverslip, add a drop of a bead solution that has been diluted in ethanol and contains six-micron diameter polystyrene beads. Once the solvent evaporates, check the spacing of the beads using a bright-field microscope equipped with a 20x objective. Ensure that the individual beads are well separated.

Then, dip a micropipette tip or a toothpick into a well-mixed epoxy glue, and transfer a small amount of the glue to three separate spots with successive gentle touches on the right side but close to the center of the coverslip, as shown here. Next, mount a cleaned, tipless cantilever to the AFM scanning head, and calibrate it in air with a clean surface to obtain the spring constant. Then, position the cantilever tip over the left boundary of the last epoxy glue spot, and slowly bring the cantilever close to the glue using small step sizes on the stepper motors.

Once contact has been made with the glue, swiftly pull the cantilever laterally by moving the AFM scanning head backward. Remove any excess glue by rubbing it off against the glass, leaving only a tiny amount of the glue at the very end of the tip. Now, move the cantilever tip on top of a well-isolated bead.

Approach the single bead slowly, and make a firm contact with it in the range of two to five nanonewtons for about 10 seconds. While making contact, use fine-tip adjustment to position the bead at the very end of the tip. Retract the tip at the end of the contact.

If the bead disappears from the original focal plane, a successful adhesion event has occurred. Finally, demount the bead-modified cantilever carefully, and store it in a cantilever box overnight for the epoxy to fully cure. Here are typical force-distance curves from the binding interaction between a single T cell and a single dendritic cell over the course of one approach-retract cycle.

The light red curve is the extension curve, and the dark red one is the retraction curve. The minimum value in the curve gives a measure of the maximum adhesion force between the T cell and dendritic cell, and the area under the curve represents the work required to separate them. The sharp and stepwise rupture events can be interpreted as membrane tethers being pulled out from the cell surface due to the strong binding at the cell-cell interface and then breaking discretely under the continuous pulling.

Here, conventional T cell binding to dendritic cells is shown in gray, and regulatory T cell binding to dendritic cells is shown in blue. The adhesion forces between a conventional T cell and a regulatory T cell recognizes peptide antigens presented by antigen-presenting cells, whereas regulatory T cells are suppressor T cells that shut down conventional T cell-mediated immunity toward the end of an immune reaction. This schematic of a fluorescently labeled, phagocytic RAW264.7 cell shows the cell being approached with a single naked, six-micron diameter, polystyrene bead.

Upon engaging the bead, membrane PIP2 is sorted at the contact site, near b, which then induces membrane recruitment of moesin, resulting in a phagocytic event. In addition to the procedure described here, AFM-based single-cell force spectroscopy can be used together with fluorescence imaging to study immune cell activation in real time at the single-cell level. Combined fluorescence techniques, this method allows for addressing more complex cellular processes in real time, such as the formation of an immune synapse between dendritic cell and T cell pair.