This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering (BES-MSE). Kinesin synthesis and fluorescence microscopy were performed through a user project (ZIM) at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science.

1. Preparation of stock microtubule solutions
CAUTION: Safety goggles, gloves and a lab coat should always be worn throughout the protocol.
<ol>
	<li>Prepare 5x BRB80 buffer: add 24.19 g of PIPES (piperazine-N,N&#8242;-bis[2-ethanesulfonic acid]) and 0.38 g of EGTA (ethylene glycol-bis[&#946;-aminoethyl ether]-N,N,N&#8242;,N&#8242;-tetraacetic acid) to a 1 L glass bottle. Add 1 mL of 1 M MgCl2 and adjust the pH to 6.9 with KOH. Add deionized water to bring the solution to a 500 mL final volume.</li>
	<li>Prepare 100 mM stock of GTP solution: weigh 52 mg of GTP and suspend in 1 mL of distilled water. Divide 100 mM solution into 20 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare GPEM solution: mix 200 &#181;L of 5x BRB80, 10 &#181;L of 100 mM GTP solution, 100 &#181;L of 100% glycerol, and 600 &#181;L of deionized water. Divide GPEM solution in 100 &#181;L aliquots and store in -20 &#176;C.&#160;</li>
	<li>Prepare microtubule solution by reconstituting vials of commercially available, lyophilized tubulin (one vial each of biotinylated, fluorescently labeled, and unlabeled) in cold (4 &#176;C) GPEM solution to a stock concentration of 5 mg/mL.</li>
	<li>Perform microtubule polymerization by mixing 4 &#181;L of biotinylated tubulin, 4 &#181;L of fluorescently labeled tubulin, and 24 &#181;L of unlabeled tubulin (all at 5 mg/mL concentrations) to create a ratio of 1:1:6 at a final volume of 32 mL. Keep it on ice. Divide the tubulin mixture into 2 &#181;L aliquots and store at -80 &#176;C until needed. 
	NOTE: Efficient polymerization requires that the concentration of tubulin is equal to or above the critical concentration (5 mg/mL)14. Here, selection of the tubulin ratio is optimized for a sufficient concentration biotinylated tubulin to efficiently bind streptavidin and GUVs, as well as a sufficient concentration of fluorescent tubulin for microscopic characterization.</li>
</ol>
2. Preparation of giant unilamellar vesicles (GUVs)
<ol>
	<li>Agarose film preparation 
	NOTE: This protocol is adapted from Greene et al.15.
	<ol>
		<li>Prepare a 1% w/v solution by mixing 1 g of agarose in 100 mL of deionized water in 250 mL Erlenmeyer flask. Use a standard microwave to heat the agarose solution for 1&#8211;2 min. 
		NOTE: The solution will become translucent once the agarose is completely dissolved. Allow the solution to cool to 65&#8211;75 &#176;C before use.</li>
		<li>Use a cut 1,000 &#181;L pipette tip to pipette 300&#8211;400 &#181;L of agarose solution onto a 25 mm x 25 mm glass coverslip. While holding the edge of the coverslip with gloved fingers, use another 1,000 &#181;L pipette tip to spread the melted agarose evenly across the coverslip. 
		NOTE: Maintaining the agarose at 65&#8211;75 &#176;C will allow for efficient spreading on the coverslip surface.</li>
		<li>Dry the agarose-coated coverslips in a 37 &#176;C incubator for at least 2 h, at which point the agarose will become transparent. Store the coverslips by placing the agarose coated surface facing upwards on a clean surface such as lint-free paper or wax-based film at room temperature (RT).</li>
	</ol>
	</li>
	<li>Lipid formulation
	<ol>
		<li>Retrieve lipids dissolved in chloroform from a -20 &#176;C freezer and place them in a chemical fume hood until they reach RT.</li>
		<li>Calculate the volume of lipid stock of each component lipid needed for the formulation using following formula: 
		<img alt="Equation 1" src="/files/ftp_upload/60899/60899eq1.jpg" /> 
		Where: Vlipid is the volume of lipid to use, mol % is the molar percentage of the lipid component, n is the total number of moles of lipid used in the formulation, and M is the concentration of lipid in molar units. 
		NOTE: For example, if using a formulation containing 45 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with a stock solution concentration of 12.72 mM, and 1 micromole of total lipids in the formulation, the volume of DOPC stock used in the formulation would be: 
		<img alt="Equation 2" src="/files/ftp_upload/60899/60899eq2.jpg" /></li>
		<li>Mix the lipids together at the calculated ratio in a glass vial in the chemical fume hood.</li>
		<li>Pipette 30 &#181;L of lipid solution onto the agarose-coated coverslips on a preheated hot plate that is set above the melting point of the saturated lipid component of the formulation (e.g., 50 &#176;C hot plate for a lipid with Tm of 40 &#176;C).</li>
		<li>Spread the solution across the agarose film in a circular motion using the long edge of an 18 G needle to until the chloroform has evaporated and a uniform layer of lipid has formed. Hold the edge of the coverslip with gloved fingers while performing this step. 
		NOTE: Care should be taken not to damage the agarose layer with the needle.</li>
		<li>Place the coverslip with agarose layer and lipid film in an aluminum foil-covered Petri dish, film side facing upwards, and place the Petri dish in a vacuum desiccator for at least 2 h to remove residual solvent.</li>
	</ol>
	</li>
	<li>In the meantime, prepare 560 mM sucrose solution by mixing 1.92 g of sucrose with 10 mL of deionized water. 
	NOTE: The concentration of the sucrose solution depends on the osmolarity of the buffer GUVs are diluted in. Typically, the osmolarity of the sucrose solution should be no more than 10% larger than the buffer the GUVs will be diluted in, specifically the motility buffer (see step 3.11).</li>
	<li>GUV formation
	<ol>
		<li>Adhere an adhesive chamber to the coverslip coated with the lipid film by gently pressing the adhesive chamber onto the lipid-coated coverslip with the lipid film facing upwards, ensuring that a tight seal is formed.</li>
		<li>Add 400 &#181;L of 560 mM sucrose solution (prepared in step 2.3) to the chamber.&#160;</li>
		<li>Place the coverslip into the humidity chamber and close the lid.</li>
		<li>Place the humidity chamber on a preheated hot plate set above the melting point of the saturated lipid component of the formulation.</li>
		<li>Allow vesicles to form for &#8805;1 h before recovery. 
		NOTE: Vesicle formation may be checked with fluorescence microscopy with a 40x air objective lens.</li>
	</ol>
	</li>
</ol>
3. Preparation of motility assay stocks and reagents
<ol>
	<li>Preparation of casein stock
	<ol>
		<li>Add 3 g of dry casein to a 50 mL conical centrifuge tube, then add 30 mL of 1x BRB80 and rotate until the solution becomes viscous. Centrifuge the tube at 15,000 x g for 30 min.</li>
		<li>Transfer the supernatant into another 50 mL conical centrifuge tube and discard the pellet. Filter the solution through a 1 &#181;m syringe filter, collecting the solution in a 50 mL conical vial. Filter the solution through a 0.2 &#181;m filter, collecting the solution in a 50 mL conical vial.</li>
		<li>Determine the protein concentration by measuring the absorbance at 280 nm using a UV-Vis spectrophotometer and quartz cuvette.</li>
		<li>Calculate the casein concentration in mg/mL using the following formula: 
		<img alt="Equation 3" src="/files/ftp_upload/60899/60899eq3.jpg" /></li>
		<li>Dilute the solution to 20 mg/mL in 1x BRB80, divide into 100 &#181;L aliquots, and store at&#160;-20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare glucose oxidase (2 mg/mL) stock solution by mixing 2 mg of glucose oxidase with 1 mL of 1x BRB80. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare catalase (0.8 mg/mL) stock solution by mixing 0.8 mg of catalase with 1 mL of 1x BRB80. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 2 M glucose solution by suspending 0.3 g of D-glucose in 1 mL of deionized water. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 100 mM DTT stock by suspending 0.015 g of DTT in 1 mL of deionized water. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 100 mM Mg-ATP stock by suspending 0.055 g of disodium ATP in 1 mL solution of 100 mM MgCl2. Divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 100 mM Mg-AMP-PNP solution by suspending out 0.055 g of AMP-PNP in a 1 mL solution of 100 mM MgCl2, then divide into 100 &#181;L aliquots and store at -20 &#176;C.</li>
	<li>Prepare 100 mM Trolox solution by adding 25.03 mg of Trolox to 1 mL of methanol and store at -20 &#176;C.</li>
	<li>Prepare 10 mg/mL streptavidin solution by adding 1 mg of streptavidin to 100 &#181;L of BRB80, then divide into 2 &#181;L aliquots and and store at -80&#1755; &#176;C.</li>
	<li>Prepare BRB90CAT by mixing 200 &#181;L of 5x BRB80, 20 &#181;L of casein solution, 10 &#181;L of MgATP solution, 10 &#181;L of Trolox, 5 &#181;L of paclitaxel solution, and 765 &#181;L of DI water. Store at 4 &#176;C.</li>
	<li>Prepare the motility solution by mixing 192 &#181;L of BRB80CAT, 2 &#181;L of D-glucose solution, 2 &#181;L of glucose oxidase solution, 2 &#181;L of DTT solution, and 2 &#181;L of catalase solution. Store at 4 &#176;C.</li>
	<li>Prepare a 1 &#181;M kinesin solution by diluting the stock kinesin solution in BRB80CAT (e.g., 2 &#181;L of 50 mM kinesin solution in 98 &#181;L of BRB80CAT) and store at 4 &#176;C. &#160;</li>
	<li>Prepare a 10 &#181;g/mL microtubule solution by diluting 10 &#181;L of stabilized microtubules in 90 &#181;L of room temperature BRB80CAT. Store at RT.</li>
	<li>Prepare a 10 &#181;g/mL streptavidin solution by adding 0.1 &#181;L of 10 mg/mL streptavidin solution in 99.9 &#181;L of motility solution. Store at 4 &#176;C.</li>
	<li>Prepare a 12x GUV solution by diluting 5 &#181;L of GUV stock into 55 &#181;L of motility solution. Store at 4 &#176;C.</li>
	<li>Polymerization of tubulin into microtubules
	<ol>
		<li>Collect a previously prepared 2 &#181;L aliquot of tubulin from the -80 &#176;C freezer (prepared in step 1.5) and place into a 37 &#176;C water bath for 30 min.</li>
		<li>Prepare a 2 mM paclitaxel solution by adding 1.71 mg of paclitaxel in 1 mL of anhydrous DMSO, divide into 10 &#181;L aliquots, and store at -20 &#176;C.</li>
		<li>Freshly prepare BRB80T by mixing 99.5 &#181;L of 1x BRB80 with 0.5 &#181;L of 2 mM paclitaxel. &#160;Warm 100 &#181;L of BRB80T to 37 &#176;C in the water bath.</li>
		<li>After 30 min, add the 100 &#181;L of BRB80T to the tubulin aliquot to stabilize the microtubules. Store at RT.</li>
	</ol>
	</li>
</ol>
4. Gliding motility assay (GMA)
<ol>
	<li>Prepare a flow chamber by affixing two strips of double-sided tape separated by 5 mm onto a glass slide. Repeat this process until three layers of tape comprise each strip.</li>
	<li>Place a coverslip on top of the tape, then press down gently on the coverslip/tape interface with a tweezer or pen to ensure sufficient adhesion. 
	NOTE: The channel should be 5 mm in width by 25 mm in length by 300 mm in height.</li>
	<li>Pipette 30 &#181;L of 1 mm kinesin solution (prepared in step 3.12) into the flow cell and let it incubate for 5 min. 
	NOTE: The casein forms a bilayer on the surface of the coverslip/glass slide and facilitates attachment of the kinesin tail to the surface.</li>
	<li>Pipette 30 &#181;L of 10 &#181;g/mL microtubule solution (prepared in step 3.13) into the flow cell, using a laboratory wipe pressed gently against the opposite end of the flow channel to facilitate solution exchange. Incubate for 5 min.</li>
	<li>Wash the flow cell 1x&#8211;3x with 1x motility solution at RT (prepared in step 3.11). &#160; 
	NOTE: Fluorescence microscopy using a 40x air objective may be used at this point to confirm microtubule attachment and motility. Microtubules appear as fluorescent filaments (tens of microns in length) moving (gliding) across the surface at ~0.5 &#956;m/s (Figure 1).</li>
	<li>Pipette 30 &#181;L of 10 &#181;g/mL streptavidin solution (prepared in step 3.14) into the flow cell using a laboratory wipe pressed gently against the opposite end of the flow channel to facilitate solution exchange. Incubate for 10 min.</li>
	<li>Flow 30 &#181;L of 12x GUV solution (prepared in step 3.15) into the flow using a laboratory wipe pressed gently against the opposite end of the flow channel to facilitate solution exchange. Incubate for 30 min.</li>
	<li>Add 2 &#181;L of 100 mM AMP-PNP solution (prepared in step 3.7) to stop motility, then seal the chamber with sealant.</li>
</ol>
5. LNT network characterization
<ol>
	<li>Transfer the flow chamber to an inverted microscope for imaging.</li>
	<li>Choose the appropriate filter set based on the wavelengths of the fluorescent lipids or tubulin used. For example, when using Texas Red-labeled lipids, use a 560 nm/25 nm excitation filter and 607 nm/36 nm emission filter.</li>
	<li>Use a 100x oil objective to focus on the surface of the coverslip.</li>
	<li>Image the LNT networks using fluorescence microscopy. 
	NOTE: LNTs are linear structures extruding from the larger vesicles. LNTs much smaller than GUVs and have weaker fluorescence signals. Thus, the exposure and contrast must be adjusted accordingly to image LNTs. These adjustments also lead to overexposure of the GUVs, and thus, it is recommended that the lamellarity and phase separation in GUVs be characterized independently.</li>
	<li>Focus the microscope on a network of interest and take a standard or tiled image.</li>
	<li>Adjust the exposure time and neutral density filters to image the LNTs and minimize saturated exposure of the GUVs. Acquire images both in red and green channels. 
	NOTE: Here, the red channel permits visualization of the Texas-Red lipids, while the green channel permits visualization of the microtubules (e.g., Oregon Green lipids and HiLyte488 dyes).</li>
	<li>Create a composite image by overlaying the red and green channels (Figure 1).</li>
	<li>Characterization of LNT networks by measuring LNT length
	<ol>
		<li>Open the acquired images using an image analysis software such as ImageJ.</li>
		<li>Calibrate the scale for the microscope by using the set scale feature, fill in the pixels to the mm conversion factor, and click OK. 
		NOTE: The conversion factor is dependent on the microscope, objective lens, and camera, and it can be obtained using a microscope calibration slide. It is generally expressed in pixels/mm.</li>
		<li>Use the Multipoint line tool to measure the length of the nanotubes starting from the parent GUV. Hold Ctrl +M to measure the length.</li>
		<li>Continue measuring the lengths of individual tubes following the steps above. The image processing tool will save each new measurement in the results window.</li>
		<li>Hold Ctrl + D after drawing each line to keep track of which tubes have been measured.</li>
	</ol>
	</li>
	<li>Measuring LNT thickness (Figure 2)
	<ol>
		<li>Open the image in ImageJ and select the Threshold feature under the Image tab.</li>
		<li>Click Apply to apply the threshold.</li>
		<li>Draw a rectangle of known length over the desired tube (black pixels have a value of 0, and red pixels have a value of 255).</li>
		<li>Measure the integrated density of the area.</li>
		<li>Divide the density by the length (in pixels) of the LNT to obtain the thickness (in pixels). 
		NOTE: The thickness measurements may only be compared across images when the imaging settings and threshold are set identically.</li>
	</ol>
	</li>
	<li>Determining the lipid partitioning in nodes of LNTs (Figure 3)
	<ol>
		<li>Open the image in ImageJ.</li>
		<li>Use the Line tool to draw a line over the desired node.</li>
		<li>Measure the node intensity in both the Oregon Green and Texas Red channels.</li>
		<li>Move the line to the LNT and measure the LNT intensity in both the Oregon Green and Texas Red channels.</li>
	</ol>
	</li>
</ol>

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Sandia National Laboratories is a multi-mission laboratory managed and operated by National Technology &#38; Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. DOE&#8217;s National Nuclear Security Administration under contract DE-NA-0003525. This paper describes objective technical results and analysis. Any subjective views or opinions that might be expressed in the paper do not necessarily represent the views of the U.S. Department of Energy or the United States Government.

LNT networks are a useful tool for in vitro studies to membrane properties and the transport of biomolecules such as transmembrane proteins. Moreover, using complex lipid formulations to fabricate LNT networks enables more biologically relevant studies. Other fabrication studies have used either 1) simple lipid formulations and multilamellar vesicles or 2) more cumbersome motility techniques to fabricate networks from GUVs comprised of complex lipid formulations. The method described here enables the efficient fabricatio...

The fabrication of lipid nanotube (LNT) networks is of increasing interest for in vitro examination of nonequilibrium lipid structures1,2,3. Cells use lipid tubules for the diffusive transport of proteins4 and nucleic acids5 as well as cell-to-cell communication6,7. The endoplasmic reticulum and Golgi apparatus are particularly interesting, as these membrane-bound organelles are the primary locations for lipid and protein synthesis as well as transport of these integral biomolecules within the cytoplasm of a cell8,9. The membranes of these organelles are comprised of multiple lipid species including sphingolipids, cholesterol, and phospholipids10 that ultimately help define their functionality. Thus, to more closely replicate and study these organelles, in vitro LNTs must be fabricated from vesicles with increasingly complex lipid formulations11.
Giant unilamellar vesicles (GUVs) are used pervasively for studying lipid membrane behavior because they can be reliably synthesized with complex formulations that include cholesterol, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)12,13. Described here is a method to fabricate LNTs from GUVs with varying lipid formulations using the gliding motility assay (GMA), in which LNTs are extruded based on the work performed by kinesin motors and microtubule filaments acting on GUVs. In this system, kinesin motor proteins adsorbed to a surface propel biotinylated microtubules, converting chemical energy from the hydrolysis of ATP into useful work (specifically, the extrusion of LNTs from biotinylated vesicles)11. The resulting LNT network provides a model platform to study effects of the differences in lipid phases on changes in LNT morphology.
Briefly, kinesin motor proteins are introduced into a flow chamber in a solution containing casein, which enables the adsorption of the motors onto the glass surface of the chamber. Next, biotinylated microtubules in a solution containing ATP flow through the chamber and are allowed to bind to the kinesin motors and begin motility. A streptavidin solution is then introduced into the chamber and allowed to bind non-covalently to the microtubules. Finally, GUVs containing a biotinylated lipid are introduced into the chamber and bind to the streptavidin-coated microtubules, then extrude LNTs to form large-scale networks over the course of 15&#8211;30 min. This method produces large, branched LNT networks using standard laboratory equipment and reagents at a low cost11.

<table border="0" cellpadding="0" cellspacing="0" style="width:1071px;" width="1071">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="64">
			<td height="64" style="height:65px;width:328px;">100x/1.4 Numerical Aperture Oil Immersion Objective</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;">1-U2B836</td>
			<td style="width:520px;">Olympus UPlanSApo 100x/1.40 Oil Objective Infinity Corrected, RMS Thread 
			Working Distance 0.12mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">3.0 ND Filter</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Neutral Density Filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">AMP-PNP</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2647</td>
			<td>(&#946;,&#947;-imidoadenosine 5&#8242;-triphosphate)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">ATP</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7699</td>
			<td>Adenosine 5&#39;-triphosphate disodium salt hydrate BioXtra</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:328px;">Brightline Pinkel DA/FI/TR/Cy5/Cy7-5X-A000 filter set</td>
			<td style="width:104px;">Semrock</td>
			<td style="width:119px;">LED-DA/FI/TR/Cy5/Cy7-5X-A-000</td>
			<td style="width:520px;"> 
			BrightLine Pinkel filter set, optimized for DAPI, FITC, TRITC, Cy5 &#38; Cy7 and other like fluorophores, illuminated with LED-based light sources</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Casein</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">22090</td>
			<td>Casein hydrolysate for microbiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Catalase</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">C9322</td>
			<td>Catalase from Bovine Liver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Chloroform</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">288306</td>
			<td>Chloroform anhydrous contains 0.5-1.0% ethanol as stabilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Cholesterol</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">700000P</td>
			<td>cholesterol (ovine wool, &#62;98%) (powder)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">D-Glucose</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7021</td>
			<td style="width:520px;">D-(+)-Glucose powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, &#8805;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DOPC</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">850375C</td>
			<td>1,2-Dioleoyl-sn-glycero-3-phosphocholine (in chloroform)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DOPE-Biotin</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">870282C</td>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DPPC</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">850355P</td>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (powder)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DPPE-Biotin</td>
			<td style="width:104px;">Avanti</td>
			<td style="width:119px;">870285P</td>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DTT</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">43816</td>
			<td>DL-Dithiothreitol solution 1 M</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">EGTA</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4378</td>
			<td style="width:520px;">EGTA, Egtazic acid, Ethylene-bis(oxyethylenenitrilo)tetraacetic acid, Glycol ether diamine tetraacetic acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">Glucose Oxidase</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">G6125</td>
			<td style="width:520px;">Glucose Oxidase from Aspergillus niger 
			Type II, &#8805;10,000 units/g solid (without added oxygen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Glycerol</td>
			<td style="width:104px;">Fisher</td>
			<td style="width:119px;">G33</td>
			<td>Glycerol (Certified ACS), Fisher Chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">GTP</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8877</td>
			<td>Guanosine 5&#8242;-triphosphate sodium salt hydrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">IX-81 Olympus Microscope</td>
			<td style="width:104px;">Olympus</td>
			<td style="width:119px;">N/A</td>
			<td>IX81 Inverted Microscope from Olympus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">KOH</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">1050121000</td>
			<td>Potassium Hydroxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Magnesium Chloride</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">M1028</td>
			<td>1.00 M magnesium chloride solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Orca Flash 4.0 Digital Camera</td>
			<td style="width:104px;">Hamamatsu</td>
			<td style="width:119px;">C13440-20CU</td>
			<td>ORCA-Flash 4.0 V3 Digital CMOS camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Oregon Green-DHPE</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">O12650</td>
			<td>Oregon Green 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Paclitaxel</td>
			<td style="width:104px;">ThermoFisher</td>
			<td style="width:119px;">P3456</td>
			<td>Paclitaxel (Taxol Equivalent) - for use in research only</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">PIPES</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6757</td>
			<td style="width:520px;">1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&#8242;-bis(2-ethanesulfonic acid)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">Texas Red-DHPE</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">T1395MP</td>
			<td style="width:520px;">Texas Red 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, 
			Triethylammonium Salt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Trolox</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">238813</td>
			<td>(&#177;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tubulin, Biotin</td>
			<td style="width:104px;">Cytoskeleton</td>
			<td style="width:119px;">T333P</td>
			<td>Tubulin protein (biotin) porcine brain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tubulin, Hy-Lite 488</td>
			<td style="width:104px;">Cytoskeleton</td>
			<td style="width:119px;">TL488M</td>
			<td>Tubulin protein (fluorescent HiLyte 488) porcine brain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tubulin, Unlabeled</td>
			<td style="width:104px;">Cytoskeleton</td>
			<td style="width:119px;">T240</td>
			<td>Tubulin protein porcine brain</td>
		</tr>
	</tbody>
</table>

fabricating multi-component lipid nanotube networks using the gliding kinesin motility assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

LNT networks (Figure 4) were fabricated using the described protocol, which uses the work performed by kinesin transport of microtubules to extrude LNTs from GUVs. Briefly, GUVs were prepared using agarose gel rehydration using sucrose solution, and microtubules were polymerized in GPEM solution and stabilized in BRB80T. Next, kinesin motors were introduced into a flow cell forming an active layer&#160;of motors on the surface of the coverslip. Microtubules were then introduced and a strepta...

Watch this Scientific Journal Video about Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay at JoVE.com

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

fabricating-multi-component-lipid-nanotube-networks-using-gliding

Research

JoVE Journal

Bioengineering

1.6K Views.  Sandia National Laboratories. This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Video: Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 &#181;m biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

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 emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

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

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &amp;#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

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 processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

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

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...