Name: ligand nano-cluster arrays in a supported lipid bilayer
Uploaded: 2017-04-23T14:00:00
Description: Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

We thank Laurent Limozin, Pierre Dillard and Astrid Wahl for continuing fruitful discussions about cellular applications. We also thank Frederic Bedu from PLANETE cleanroom facility for his help with SEM observations. This work was partially funded by the European Research Council via grant No. 307104 FP/2007-2013/ERC.

1. Cleaning Glass Cover-slides and Observation Chambers
<ol>
	<li>Arrange the glass cover-slides on a multi-slide tray made of an inert material like Polytetrafluoroethylene (PTFE).</li>
	<li>Immerse the tray with slides and the observation chamber in a surfactant solution (any product recommended for cleaning quartz cuvettes is suitable).</li>
	<li>Using an ultrasonic bath, ultra-sonicate in the surfactant solution for 30 min at room temperature (between 20 and 30 &#176;C).</li>
	<li>Rinse 5 times with ultrapure water...

<ol>
	<li>Alberts, B., Johnson, A., Lewis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Cell. , (2002).</li><li>Varma, R., Campi, G., Yokosuka, T., Saito, T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Cell+Receptor-Proximal+Signals+Are+Sustained+in+Peripheral+Microclusters+and+Terminated+in+the+Central+Supramolecular+Activation+Cluster.">T Cell Receptor-Proximal Signals Are Sustained in Peripheral Microclusters and Terminated in the Central Supramolecular Activation Cluster.</a> Immunity. 25 (1), 117-127 (2006).</li><li>Kaizuka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+segregating+T+cell+receptor+and+adhesion+molecules+during+immunological+synapse+formation+in+Jurkat+T+cells.">Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells.</a> Proc Natl Acad Sci USA. 104 (51), 20296-20301 (2007).</li><li>Dustin, M. L., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+signaling+clusters+in+the+immune+synapse.">Receptor signaling clusters in the immune synapse.</a> Annu Rev Biophys. 41, 543-556 (2012).</li><li>Huppa, J. B., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell-antigen+recognition+and+the+immunological+synapse.">T-cell-antigen recognition and the immunological synapse.</a> Nat Rev Immunol. 3 (12), 973-983 (2003).</li><li>Grakoui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Immunological+Synapse:+A+Molecular+Machine+Controlling+T+Cell+Activation.">The Immunological Synapse: A Molecular Machine Controlling T Cell Activation.</a> Science. 285, 221-228 (1999).</li><li>Dillard, P., Varma, R., Sengupta, K., Limozin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-mediated+friction+determines+morphodynamics+of+spreading+T+cells.">Ligand-mediated friction determines morphodynamics of spreading T cells.</a> Biophys J. 107 (11), 2629-2638 (2014).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+viral+antigen+processing+generates+peptide-specific+MHC+class+I+cell-surface+clusters.">Endogenous viral antigen processing generates peptide-specific MHC class I cell-surface clusters.</a> Proc Natl Acad Sci U S A. 109 (38), 15407-15412 (2012).</li><li>Pi, F., Dillard, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-Tunable+Organic+Nanodot+Arrays:+A+Versatile+Platform+for+Manipulating+and+Imaging+Cells.">Size-Tunable Organic Nanodot Arrays: A Versatile Platform for Manipulating and Imaging Cells.</a> Nano Lett. 15 (8), 5178-5184 (2015).</li><li>Deeg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+activation+is+determined+by+the+number+of+presented+antigens.">T cell activation is determined by the number of presented antigens.</a> Nano Lett. 13 (11), 5619-5626 (2013).</li><li>Delcassian, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+ligand+spacing+influences+receptor+triggering+in+T+cells+and+NK+cells.">Nanoscale ligand spacing influences receptor triggering in T cells and NK cells.</a> Nano Lett. 13 (11), 5608-5614 (2013).</li><li>Matic, J., Deeg, J., Scheffold, A., Goldstein, I., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+tuning+and+efficient+T+cell+activation+with+stimulatory+aCD3+nanoarrays.">Fine tuning and efficient T cell activation with stimulatory aCD3 nanoarrays.</a> Nano Lett. 13 (11), 5090-5097 (2013).</li><li>Dillard, P., Pi, F., Lellouch, A. C., Limozin, L., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-clustering+of+ligands+on+surrogate+antigen+presenting+cells+modulates+T+cell+membrane+adhesion+and+organization.">Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization.</a> Integr Biol. 8 (3), 287-301 (2016).</li><li>Pi, F., Dillard, P., Limozin, L., Charrier, A., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometric+protein-patch+arrays+on+glass+and+polydimethylsiloxane+for+cell+adhesion+studies.">Nanometric protein-patch arrays on glass and polydimethylsiloxane for cell adhesion studies.</a> Nano lett. 13 (7), 3372-3378 (2013).</li><li>Fenz, S. F., Merkel, R., Sengupta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+and+intermembrane+distance:+case+study+of+avidin+and+E-cadherin+mediated+adhesion.">Diffusion and intermembrane distance: case study of avidin and E-cadherin mediated adhesion.</a> Langmuir. 25 (2), 1074-1085 (2009).</li><li>Sengupta, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+tissue+surfaces+by+supported+membrane+coupled+ultra-thin+layer+of+hyaluronic+acid.">Mimicking tissue surfaces by supported membrane coupled ultra-thin layer of hyaluronic acid.</a> Langmuir. 19 (5), 1775-1781 (2003).</li><li>Taylor, Z. R., Keay, J. C., Sanchez, E. S., Johnson, M. B., Schmidtke, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independently+controlling+protein+dot+size+and+spacing+in+particle+lithography.">Independently controlling protein dot size and spacing in particle lithography.</a> Langmuir. 28 (25), 9656-9663 (2012).</li><li>Massou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+ordered+topographical+and+chemical+nano-features+from+anodic+alumina+templates.">Large scale ordered topographical and chemical nano-features from anodic alumina templates.</a> Appl. Surf Sci. 256 (2), 395-398 (2009).</li><li>Selhuber-Unkel, C., Lopez-Garcia, M., Kessler, H., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperativity+in+adhesion+cluster+formation+during+initial+cell+adhesion.">Cooperativity in adhesion cluster formation during initial cell adhesion.</a> Biophys J. 95 (11), 5424-5431 (2008).</li><li>Arnold, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cell+polarization+and+migration+by+a+gradient+of+nanoscale+variations+in+adhesive+ligand+spacing.">Induction of cell polarization and migration by a gradient of nanoscale variations in adhesive ligand spacing.</a> Nano Lett. 8 (7), 2063-2069 (2008).</li><li>Cavalcanti-Adam, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+spreading+and+focal+adhesion+dynamics+are+regulated+by+spacing+of+integrin+ligands.">Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands.</a> Biophys J. 92 (8), 2964-2974 (2007).</li><li>Schvartzman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanolithographic+Control+of+the+Spatial+Organization+of+Cellular+Adhesion+Receptors+at+the+Single-Molecule+Level.">Nanolithographic Control of the Spatial Organization of Cellular Adhesion Receptors at the Single-Molecule Level.</a> Nano Lett. 11 (3), 1306-1312 (2011).</li><li>Mossman, K., Groves, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterned+supported+membranes+as+tools+for+quantitative+studies+of+the+immunological+synapse.">Micropatterned supported membranes as tools for quantitative studies of the immunological synapse.</a> Chem.Soc.Rev. 36 (1), 46-54 (2007).</li><li>Furlan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatase+CD45+both+positively+and+negatively+regulates+T+cell+receptor+phosphorylation+in+reconstituted+membrane+protein+clusters.">Phosphatase CD45 both positively and negatively regulates T cell receptor phosphorylation in reconstituted membrane protein clusters.</a> J Biol Chem. 289 (41), 28514-28525 (2014).</li><li>Hsu, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+mobility+modulates+immunological+synapse+formation+and+T+cell+activation.">Ligand mobility modulates immunological synapse formation and T cell activation.</a> PloS One. 7 (2), e32398 (2012).</li><li>Yu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+integrin+binding+to+Arg-Gly-Asp+peptide+activates+actin+polymerization+and+contractile+movement+that+stimulates+outward+translocation.">Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation.</a> Proc Natl Acad Sci U S A. 108 (51), 20585-20590 (2011).</li></ol>

The critical steps within the protocol described above are related to the formation of the protein nano-dots or the back-filling of the space around the dots by a supported lipid bilayer. The first critical step with respect to protein nano-dots is the preparation of the bead-mask. The cleaning of the cover-slide is critical. The slides need to be either cleaned with a detergent solution that is recommended for cleaning quartz cuvettes, or with oxygen plasma. Other cleaning techniques like immersion in ethanol or iso-pro...

Cell adhesion takes place through specialized cell adhesion molecules (CAMs), proteins present on the cell membrane that are capable of binding to their counterpart on the extra cellular matrix or on another cell. On adhered cells, most adhesion molecules including the ubiquitous integrin and cadherin, are found in the form of clusters 1. The interaction of T lymphocytes (T cells) with antigen presenting cells (APCs) provides a particularly striking illustration of the importance of receptor clusters formed at the interface between the two cells &#8212; often called an immunological synapse. Upon forming the first contact with the APC, T cell receptors (TCRs) on the surface of the T cell form micron scale clusters that serve as signaling platforms 2,3,4, and are eventually centralized to form a larger central supramolecular cluster (cSMAC) 5,6,7. Recently, it was shown that on the APC side, the ligands of the TCR are also clustered 8.
In the context of T cell-APC interaction, the deployment of hybrid systems, where the APC is mimicked by an artificial surface functionalized with relevant proteins, has greatly advanced our understanding of the synaptic interface 2,3,4,5,6,7. In this context, it is highly relevant to design APC mimetic surfaces that capture one or more aspects of the target cell. For example, if ligands are grafted on supported lipid bilayers, they can diffuse in the plane of the bilayer, mimic the situation on the APC surface and at the same time allow the formation of the cSMAC 6,7. Similarly, the clusters on the APC have been mimicked by creating islands of ligands in a sea of polymers 9,10,11,12,13,14. However, these two features have so far not been combined.
Here we describe a novel technique to create nano-dots of anti-CD3 (an antibody that targets the TCR complex) surrounded by a lipid bilayer with diffusing lipids. The bilayer is deposited using Langmuir-Blodgett/Langmuir-Schaefer technique 7,15,16 and if desired, could be functionalized with a specific protein &#8212; for example, the ligand of the T cell integrin (called ICAM1). In addition, the anti-CD3 protein dots could be replaced with another antibody or CAM. While we have chosen the proteins for future use as platform for T cell adhesion studies, the strategy detailed here can be adapted for any protein and even DNA.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass coverslips</td>
			<td>Assistent, Karl Hecht KG&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Observation chamber</td>
			<td>Home made</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline surfactant concentrate (Hellmanex)</td>
			<td>Hella Analytics</td>
			<td>9-307-011-4-507</td>
		</tr>
		<tr>
			<td>Ultra-sonicator</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Labbox</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystallizer&#160;</td>
			<td>Shott</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidine</td>
			<td>Thermo Fischer Scientifique</td>
			<td>84607</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Sigma-aldrich</td>
			<td>P3813</td>
		</tr>
		<tr>
			<td>Water MQ&#160;</td>
			<td>ELGA, Veolia France</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica beads</td>
			<td>Corpuscular Inc</td>
			<td>147114-10</td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma-aldrich</td>
			<td>A3648</td>
		</tr>
		<tr>
			<td>BSA-Biotin</td>
			<td>Sigma-aldrich</td>
			<td>A8549</td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
		</tr>
		<tr>
			<td>Dansyl-PE</td>
			<td>Avanti Polar Lipids</td>
			<td>810330C</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-aldrich</td>
			<td>650471</td>
		</tr>
		<tr>
			<td>Gastight syringe&#160;</td>
			<td>Dominique Dustcher , France</td>
			<td>74453</td>
		</tr>
		<tr>
			<td>Film balance</td>
			<td>NIMA</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss, Germany</td>
			<td>TIRF-III system</td>
		</tr>
		<tr>
			<td>Aluminium Target&#160;</td>
			<td>Kurt J. Lesker Compagny, USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Radio Frequency Magnetron sputtering Syst&#232;me&#160;</td>
			<td>modified SMC 600 tool by ALCATEL , France</td>
			<td></td>
		</tr>
	</tbody>
</table>

ligand nano-cluster arrays in a supported lipid bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

The fluorescence images were analyzed to measure the spacing and size of the dots. Typical spacing was found to be 1,900 &#177; 80 nm and typical dot-size was 600 &#177; 100 nm (Figure 1g). The spacing is set by the size of the beads used for the mask. The dot-size is set by the bead-size as well as deposition conditions. The SLB is deposited uniquely around the protein dots and not on them (Figure 2), with perfect complementarity between the holes seen i...

Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

The overall goal of this technique is to fabricate an array of protein nano-clusters in a supported lipid bilayer, which is compatible with sensitive surface microscopy techniques for cell biology applications. This method can help answer key questions in the field of cell biophysics, in particular, how the nano-clustering of ligands influences T cell activation and adhesion. The main advantage of this technique is that it is easily reproducible in standard biophysical lab and it is compatible with advanced surface sensitive microscopy technique, such as TIRF and RICM.Though this technique is designed to provide insight into immunology, it can be applied to other cell types with potential applications in cell biology, oncology and tissue engineering. To begin this procedure, clean the glass cover slides and observation chambers as outlined in the text protocol. Then, deposit 70 microliters of 2%silica bead suspension drop by drop onto a cover slide held at a 15 degree incline.Flip the glass 90 degrees every 15 seconds for one minute while the suspensions dries. It is critical to create a closely-packed monolayer of beads and to avoid the formation of multilayers and oil clusters. That's why the concentration and the volume of the bead solution as well as the hydrophilicity of the slide need to be optimized.After the liquid has evaporated, place the slide inside a radio frequency magnatron sputtering device onto a rotating table situated 105 millimeters away from an aluminum silicon target. After this, use a turbo-molecular pump to decrease the pressure in the deposition chamber to 2.6 times 10 to the negative fourth pascals. Introduce a pure argon atmosphere with a flux of 10 standard cubic centimeters per minute and a pressure of 0.8 pascals.Next, switch on the radio frequency power generator. After the plasma is stabilized, sputter for two minutes while keeping the shutter closed, to remove possible impurities from the target surface. Open the shutter, and allow the sputtering to continue for 60 minutes to deposit a 200-nanometer thick layer of aluminum on the slide.Then cut the flow of argon. Close the gate valve to isolate the turbo-molecular pump from the deposition chamber. After this, vent the chamber with clean nitrogen to reach ambient pressure.Recover the aluminum-coated slide from the chamber. Immerse the recovered slide in ultra-pure water at room temperature and ultra-sonicate at 50 watts and 50 hertz for 30 seconds. Next, deposit 0.5 milliters of APTES at the bottom of a desiccator.Place the glass slide on a ceramic grid. Place the grid into the desiccator. Connect the desiccator to a membrane pump.Then run the pump at maximum power for 30 minutes to generate a low vacuum. Close the valve of the desiccator, and switch off the pump. Heat to 50 degrees celsius for one hour.After this open the desiccator and collect the slide. Place the collected slide on a PTFE support. Deposit 2 milliliters of 25 micrograms per milliliter biotin dissolved in PBS.Allow the slide to rest for 30 minutes at room temperature. Then rinse 10 times with PBS. Incubate the slide in a solution of sodium hydroxide and PBS at room temperature overnight.After this, rinse the slide 10 times with ultra-pure water. After cleaning the Langmuir trough, place PTFE trays in the trough's PTFE enclosure. Then fill it with ultra-pure water.Set the measured pressure to zero millinewtons per meter. Using a gas-tight glass metal syringe, deposit 30 microliters of 1 milligram per millimeter DOPC and chloroform solution on the water's surface. Close the PTFE barrier until the desired pressure of 27 millinewtons per meter is reached to compress the lipid monolayer.Next, using a motorized clamp, dip the prepared glass slide into the PTFE enclosure. Hold the slide in the clamp perpendicular to the air-water interface. Raise the slide through the interface at a rate of 15 millimeters per minute while maintaining a constant pressure of 27 millinewtons per meter.Then, place the glass slide horizontally on the surface of the water above a PTFE tray. Next, using metal tweezers, push the slide down into the PTFE tray, immersing it in the ultra-pure water. Use the tweezers to transfer the PTFE tray containing the slide into a crystallizer filled with ultra-pure water.Transfer the coated slide underwater into an observation chamber. After this, close the chamber, ensuring that approximately 1 milliliter of water is trapped inside. Another delicate step is the assembly of the sample chamber underwater.And one should absolutely avoid contact with the air of the deposited bilayers, so to ensure this use a large volume of water and ensure that the whole assembly process can occur underwater. Remove the assembled chamber from the water, check to ensure that the chamber is watertight and free from leaks. Add 500 microliters of PBS, then remove 500 microliters of liquid from the chamber.Repeat this process 10 times to fully replace the one milliliter of ultra-pure water in the chamber with PBS. Next, introduce 100 microgram per milliliter of bovine serum albumen to the observation chamber. Incubate at room temperature for 30 minutes.After this, rinse the bilayer by removing and adding 500 microliters from the chamber, 10 times. Then, functionalize with ligands, deposit cells, and observe the proteolipidic nanopattern and SLB fluidity as outlined in the text protocol. In this protocol, a bead mask is used to create a secondary metal mask by controlled deposition of aluminum.Then, the bead mask is removed, leaving a layer of aluminum with holes. Next, an organo-amino siline, called APTES, is deposited in the vapor phase, followed by deposition of BSA-Biotin in aqueous phase. Then, aluminum is removed, revealing nanometric protein patches.Finally the inter-dot space is back-filled with a supported lipid bilayer. Epiflourescence images are then taken of the nano-dots and supported lipid bilayer. A composite image shows perfect complementarity with the lipid bilayer deposited uniquely around the protein dots, but not on them.In an epiflourescence image of a freshly-deposited supported lipid bilayer, the protein dots appear as dark spots in a bright sea of lipids. However, after continuous photo bleaching for 50 seconds, the image shows a halo inside the region delimited by the field diaphragm, indicating that the lipids are mobile. Analysis of the average intensity profile along the edge of the field diaphragm, and the decay of this intensity over time during the bleaching process, reveals that the diffusion constant is five micrometers squared per second.This technique can be done in two days if it is performed properly. Its's important to maintain very clean conditions during aluminum deposition, and to carefully calibrate the deposition curve. Following this procedure, the patterned slide can be further functionalized.For example, by adding another biomolecule in the bilayer. We use this to mimic the antigen plant-eating cells, in order to study the T cell activation. So after its development, this technique should interest diverse audiences.For example, tissue engineers may want to use it as scaffold to grow artificial tissues, and oncologists may use this as a platform for posing fundamental questions about addition of cancer cells. After watching this video you should have a good understanding of how to fabricate an area of protein nano-clusters in supported lipid bilayer, which is compatible with an advanced microscopy technique. While we use this to study T cells, we believe that this substrate have the potential to become the platform of choice to study the interaction of any kind of cells with controlled pattern proteolipidic membrane.

Research

JoVE Journal

Bioengineering

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...