Hi, I'm Mike Dustin from the Geral Institute and New York University School of Medicine. Today I'll be taking you through the procedure of forming supportive planar Biolay for study of the immunological synapse. So let's get started.
To make the lipid bilayer, we prepare a solution of 20%nickel, chelating lipid, and 80%phosphatidylcholine in the appropriate amounts in a glass tube with one to two milliliters of chloroform methanol. The first step is to evaporate the solvent under a stream of nitrogen gas in a warm 30 to 37 degrees Celsius water bath. This usually takes around 15 minutes.
Then remove any remaining solvent under high vacuum using a lyophilizer for 90 minutes. Next, dissolve the lipids in 2%N octal glucocide detergent interest saline buffer to a final concentration of 0.4 millimolar. This creates a solution of mixed detergent phospholipid mycells to form liposomes.
You dialyze this solution against three changes of tris saline to remove detergent and form liposomes. We usually work with volumes on the order of one milliliter with six millimeter diameter tubing with a cutoff around 10 kilodaltons and are careful to exclude any air bubbles while clamping the tubing. We sterile filter this solution and do the dialysis under clean conditions and handle it using 70%ethanol sterilized gloves.
This solution should be crystal clear and is stored under Argonne gas to prevent oxidation. If the solution is turbid, then you have generated multi walled vesicles and need to start again. In order to set out the experiment, we need to first determine how much ICAM one and MHC are deposited on a given surface area of nickel chelating bilayer at a given concentration of soluble protein.
To do this, we deposit bilayers on five micrometer diameter glass speeds. Incubate the glass beads with protein solutions under conditions that approximate those in the flow cells used for imaging, and then read out the density of bound protein by a flow immuno fluoro metric assay. FSI labeled antibodies with known numbers of fluorescein per molecule are used to detect the surface bound proteins and fluorescein standard beads are used to calibrate the flow micro fluorimeter.
We will establish conditions that approximate those on antigen presenting cells with 200 molecules per square micrometer of ICAM one and 0.2 to 20 molecules per square. Micrometer of I-E-K-M-C-C 91 1 0 3, complex planar bilayers form spontaneously when the liposomes fuse to glass, but only if it is properly cleaned to clean glass. We use acid piana solution, a freshly prepared mixture of sulfuric acid and 30%hydrogen peroxide, which destroys all organic material and also leaves the glass surface very hydrophilic.
When working with piranha, we wear full protective clothing, including an acid apron and heavy acid resistant gauntlets. The waste needs to be carefully segregated from organic waste and disposed of properly. To prepare the acid piranha solution, add 75 milliliters of concentrated sulfuric acid to a dry beaker and then carefully add 25 milliliters of 30%hydrogen peroxide.
The solution rapidly heats up to greater than 100 degrees Celsius, then immerse the dry cover slips in the solution for 15 minutes. Following this incubation in piranha, dunk the cover slips in purified water and then rinse them for a minute on each side. Then dry the cover slips using a vacuum source to drain off the purified water.
Allow the piranha solution to cool and add to the piranha waste container, which is left with a cap loose, well marked in the fume hood to form the bilayers. Our lab uses bi FCS two chambers, which have the advantages of integrated heating. The FCS two system is on a stainless steel base that clamps together a microfluidic manifold with a 0.5 millimeter top gasket, a micro aqueduct slide coated with indium tin oxide for heating on one surface and with a fluidic groove on the other side.
A dust-free 0.25 millimeter gasket that defines the height of the flow chamber and the piranha cleaned 40 millimeter round cover slip on which the bilayer is formed to assemble the flow cell and form planar bilayers. Place the manifold with the tubes directed up on a flat surface. Then place the 0.5 millimeter gasket with holes positioned over the fluidic tubes and make sure this is seated flat.
Gently place the micro aqueduct slide with the fluidic channels facing up and make sure it seats flat without applying any downward pressure until verifying that the holes in the slide align the microfluidic tubes. Then lay the dust free 0.25 millimeter gasket with a rectangular cutout on top of the micro aqueduct slide so that the channel is lined up with the fluidic channels and there are no major air spaces place as many as five one microliter drops of liposome suspension on the surface of the micro aqueduct slide in a pattern such that the center to center distance between each drop is 2.5 millimeters. The five liposome drops may have different compositions, but in this case we will only form two bilayers, one with no nickel chelating lipids, and one with 10%nickel chelating lipids.
Once these drops are in place, carefully place the cover slip down on the surface in one motion, clamp onto the base as quickly as possible. The liposome drops should make contact with the cover slip. This contact should not be broken since the bilayers have probably already formed and any disruptions may result in defective bilayers.
At this point, it is important to remember to mark the positions of the bilayers with a few small ink spots on the slide to aid positioning of the cover slip on the microscope. Wait 20 minutes to make sure the system has equilibrated transfer all the solutions using a 20 milliliter syringe connected to about 20 centimeters of flexible tubing and a three-way stop cock. Another port of the stop cock is then connected to the flow cell by a short length of tubing to minimize the dead volume between the stopcock and the flow cell.
Fill the syringe with heis buffered saline containing magnesium chloride, calcium chloride glucose and human serum albumin. Prime the tubing and stop cock so there are no air bubbles. Then connect this to the flow cells and push through five milliliters in one motion while watching to make sure no air bubbles pass over the bilayer.
Now you'll have your bilayers to introduce the hiss tagged proteins into the bilayer. It must first be blocked with casen and charged with nickel. Fill a one milliliter syringe with the casen nickel ion solution and connect this to the side port without trapping any air bubbles.
After injecting the solution, wait 20 minutes to allow for complete blocking. Then wash the blocking solution out with H-B-S-H-S-A from the 20 milliliter syringe. Now inject 0.5 milliliters of H-B-S-H-S-A solution containing the polyhis tagged ICAM one and IEK.
In this experiment, the ICAM one is labeled with SCI five. So the LFA one ICAM one interactions can be followed by imaging the ICAM one rearrangement in the bilayer allow 30 minutes for the protein to bind the nickel charged bilayer while the protein is adhering to the bilayer. Secure the flow cell to the inverted microscope using a custom stage insert.
Then connect the heating electronics to allow the system to equilibrate at 37 degrees Celsius. Use the marks made earlier to position the flow cell so that the objective is aligned with one of the bilayers and use interference reflection microscopy to locate the focal plane by focusing on the reflected light image of the field diaphragm. Finally, wash out the unbound ICAM one from the flow cells.
Acquire fluorescence images of the bilayer in the ICAM one channel in turf M and widefield modes. This is to confirm ICAM one binding on the nickel coated bilayer relative to the nickel free bilayer. The flow cell is now ready to receive cells.
For this demonstration, we will use a ND T-cell receptor transgenic T cells that have been transduced with a retrovirus ENC coating GFPs P zap 70, wash the cells once with H-B-S-H-S-A and then incubate the cells with an Alexa 5 68 labeled FAB to the T-cell receptor so that we can track the TCR micro clusters. After 15 minutes, dilute the cells one to 10. This dilutes the H 57 antibody enough so that it doesn't interfere with turf M imaging, but maintains saturation of the T cell receptor as the initially bound fab starts to dissociate, then inject them into the flow cell.
We image the immunological synapse in three color turf M mode. This includes the formation of the LFA one, ICAM one interaction, the formation of TCR clusters and the C smac and the sustained formation of TCR micro clusters, which recruit GFP ZAP 70. Some of these results are obvious during acquisition, whereas others will require post-processing analysis to fully appreciate.
Okay, we've just shown you how to use supported planar bilayers to study the immunological synapse. It's very important for these experiments to work that the T-cells are in very good shape and the PPS buffered saline solution is not very for long-term T-cell culture. So you wanna take the cells out of culture, put em into this media, and then use them very quickly.
If you find that you wanna do longer term experiments, there's some very good serum free media formulations that you can substitute for the he piece buffered saline. That'll keep the cells alive much longer if you want to do longer end endpoint, such as cytokine production or proliferation of the T cells. So that's it.
Thanks for watching and good luck with your experiments.
