Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

To assemble the glass-supported planar lipid bilayer, first, prepare 100 milliliters of piranha solution by mixing 30% hydrogen peroxide with sulfuric acid at a ratio of 1 to 3 in a beaker. Into this solution, place two rectangular #1.5 coverslips for 20 to 30 minutes. While the coverslips are being cleaned, retrieve one tube of previously prepared 400-micromolar DOPC lipids, and one tube of previously prepared 80-micromolar Biotin-PE lipids. Transport them on ice to the argon tank.

After deoxygenating a new microcentrifuge tube with argon, add together the DOPC and Biotin-PE at a 1 to 1 ratio. Specific volume will vary based on experimental needs, but should be, at minimum, 2 microliters each. Deoxygenate the mixture tube and the individual reagent tubes before returning the latter to the refrigerator.

After they are finished cleaning, thoroughly rinse the coverslips with distilled water. Set the coverslips out to air-dry for a few minutes. Then, withdraw 1.5 microliters of the liposome mixture, and aliquot it in a single drop into one of the lane chambers of a chamber slide. The use of two drops per lane is typical but not necessary.

Now, quickly and efficiently place the dry coverslip over the droplets. Ensure that the drops are sufficiently spaced so that they do not merge once the coverslip is placed. Furthermore, make sure that the drops remain circular and well-defined, without touching the edges of the chamber walls. Press down firmly, in between and around each lane, to ensure a watertight seal between the coverslip and slide.

Mark the positions of the drops using a marker pen. Then, pass 100 microliters of aqueous 5% casein through the chamber to block the bilayer. Try to make sure that there are no bubbles in the flow chamber. Then, inject 100 microliters of streptavidin at a concentration of 333 nanograms per milliliter into each lane.

After incubating for 10 to 15 minutes at room temperature, wash by running 3 milliliters of HEPES-buffered saline with 1% human serum albumin through each lane to remove the excess streptavidin. Add 100 microliters of biotinylated, fluorescently labeled anti-CD16 at the protein concentration previously determined, as described in the text protocol.

Following incubation in the dark for 20 to 30 minutes, wash again by running 3 milliliters of HEPES-buffered saline with 1% human serum albumin through each lane. Then, flow 100 microliters of D D-biotin at a concentration 25 nanomolar through the chamber in order to bind any excess streptavidin, and thus, eliminate the chance of nonspecific binding of streptavidin to the cells.

Next, count human NK cells, and resuspend them at a concentration of 500,000 per milliliter in HEPES-buffered saline with 1% human serum albumin. Wash the D-biotin out of the chamber with another 3 milliliters of HEPES-buffered saline with 1% human serum albumin per lane. Once the cells have been resuspended at the desired concentration, add 100 microliters to each lane. Place the chamber in a 37-degree Celsius, 5% carbon dioxide incubator for 30 to 60 minutes.

After this incubation period, fix the cells with 4% paraformaldehyde at room temperature for 10 to 20 minutes. Wash by running 3 milliliters of phosphate-buffered saline through each lane to remove the paraformaldehyde. Then, add 400 microliters of blocking buffer before incubating at room temperature for 30 minutes.

Next, stain F-actin and perforin by adding 200 microliters of diluted, fluorescently labeled phalloidin and fluorescently labeled anti-perforin monoclonal antibody. Incubate at room temperature for one hour before washing by running 3 milliliters of phosphate-buffered saline through the chamber. The chamber is ready for imaging.

To begin, turn on all necessary hardware modules of the STED microscope. Start up the image analysis software, and enable both resonant scanning and STED modules. After making these selections, wait for about three to five minutes for the software to initiate.

Click on the Configuration tab at the top of the screen. Select Laser Config. Then, turn on the white light and the STED 592-nanometer lasers. Choose the 100X objective, and align the excitation laser beam with a 592-nanometer depletion laser. Select the Laser Config module. Turn off the 592 depletion laser, and turn on the 660-nanometer depletion laser.

Place the slide upon the stage over the lens. Bring the cells bound on the demarcated bilayer region into focus using the white light lamp and the oculars. Then, return to the Acquisition tab directly to the right of the Configuration tab. Click the Switch a White Light tab, and then turn that module on, and drag the excitation laser line to the appropriate wavelength.

After selecting the desired detector and setting the detection range, click on the Sequential button in the left-hand Acquire toolbar to bring up the sequential scanning dialog in the bottom of the left-hand toolbar. This allows the user to add multiple sequences, each with a different excitation beam for a different color. Click between frames, and then, set the excitation frequency, detector, and detection range for each additional color. Once all the settings are optimized, hit Start to begin the acquisition process.