Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The overall goal of this procedure is to demonstrate how to successfully capture super resolution images of the immunological synapses of natural killer or NK cells on the supported lipid bilayer. This is accomplished by constructing a glass supported planar lipid bilayer containing antibodies against NK cell activating receptor CD 16 In chamber slides. The previously isolated NK cells are then added to the planar antibody embedded lipid bilayer, followed by staining of the cells with f actin and perforin.

The final step is imaging of the resulting NK cell synapses using the stead microscope, ultimately imaging of the NK synapse on the supported lipid bilayer. Using stead shows the NK cell synapse in enhanced detail, which allows visualization of the ultra structure of the actin cytoskeleton and perran positive litic granules positioned over regions of low F actin density. So the mainwan of this technique is that the glass support plain lipid barrier can mimic the plus membrane of tuck cell, the protein on this plain lipid barrier.

They can move laterally, which can better recapitulate of formation of very logically relevant of immuno synapse. This message can help answer key Christians in the field of immunology, allowing investigators to visualize the synapse formation in control manner with detail, which is unachievable by using commercial and microscopic approaches. A postdoc fellow painting and a graduate student grant will demonstrate the procedure 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 one to three in a beaker into this solution. Place two rectangular number 1.5 cover slips for 20 to 30 minutes while the cover slips 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 deoxy oxygenating a new micro centrifuge tube with argon. Add together the DOPC and biotin PE at a one-to-one ratio. Specific volume will vary based on experimental needs, but should be at minimum two microliters.

Each deoxy oxygenate the mixture tube and the individual reagent tubes before returning the ladder to the refrigerator. After they are finished cleaning thoroughly rinse the cover slips with distilled water. Set the cover slips out to air dry for a few minutes, then withdraw 1.5 microliters of the liposome mixture and aliquot 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 cover slip over the droplets, ensure that the drops are sufficiently spaced so that they do not merge once the cover slip 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 cover. Slip and slide mark the positions of the drops using a marker pen. Then pass 100 microliters of aqueous 5%casing 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 strept adin at a concentration of 333 nanograms per milliliter into each lane. After incubating for 10 to 15 minutes at room temperature, wash by running three milliliters of heaps buffered saline with 1%human serum albumin through each lane.

To remove the excess tritin, add 100 microliters of biotinylated fluorescently labeled anti CD 16 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 three milliliters of heaps buffered saline with 1%human serum albumin through each lane. Then flow 100 microliters of D biotin at a concentration 25 nanomolar through the chamber in order to bind any excess STR tabin and thus eliminate the chance of non-specific binding of STR tabin to the cells.

Next count human NK cells and resuspend them at a concentration of 500, 000 per milliliter in heaps buffered saline with 1%human serum albumin. Wash the D biotin out of the chamber with another three milliliters of heaps 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%paraform aldehyde at room temperature for 10 to 20 minutes. Wash by running three milliliters of phosphate buffered saline through each lane to remove the paraform aldehyde.

Then add 400 microliters of blocking buffer before incubating at room temperature for 30 minutes. Next, stain f actin and perforin by adding two microliters of diluted fluorescently labeled Phin and fluorescently labeled anti perran monoclonal antibody incubate at room temperature for one hour before washing by running three milliliters of phosphate buffered saline through the chamber. The chamber is ready for imaging to begin.

Turn on all necessary hardware modules of the stead microscope. Start up the image analysis software and enable both resonant scanning and stead 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 stead 592 nanometer lasers. Choose the 100 x 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 to 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 dialogue 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.

The triple color stead image of the NK cell synapse on glass supported planar lipid bilayer shows that anti CD 16 antibody shown in red accumulates on the lipid bilayer triggering F actin formation, and the polarization and penetration of perforin shown in blue through the f actin mesh at the focal panel. Using this combined approach, one can cleanly observe the micro clusters of fluorescently labeled anti CD 16 within the glass supported lipid bilayer, which directly mirrors the clustering of CD 16 on the NK cell. Compared to the conventional confocal image, the ring structure of CD 16 central cluster is more readily discerned in the stead image due to the depleted ambient fluorescence.

Furthermore, the ultra structure of the actin cytoskeleton is seen with significantly improved resolution. Consistent with previous observations, the perin positive lytic granules are seen positioned over regions of low F actin density. In the stead image, a crucial detail which is mostly lost in the confocal image Once mastered, this technique can be done around four hours if done properly while going through it.

It's important to remember to keep your sample deoxygenated through repeatedly displacing the oxygen with argonne, and when assembling the chamber by swiftly placing the cover slip over your sample and immersing it in the casing solution.