Name: fabricating optical-quality glass surfaces to study macrophage fusion
Uploaded: 2018-03-14T12:30:00
Description: Watch this Scientific Journal Video about Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion at JoVE.com

We wish to thank members of the Ugarova laboratory and investigators in the Center for Metabolic and Vascular Biology for helpful discussion of this work. James Faust wishes to express his gratitude to the instructors at the European Molecular Biology Laboratory Super Resolution Microscopy course in 2015. We wish to thank Satya Khuon at Janelia for help with sample preparation for LLSM. During the review and filming portions of this work James Faust was supported by a T32 Fellowship (5T32DK007569-28). The Lattice Light Sheet component of this work was supported by HHMI and the Betty and Gordon Moore Foundation. T.U. is funded by NIH grant HL63199.

Procedures that utilize animals were approved by the Animal Care and Use Committees at Mayo Clinic, Janelia Research Campus, and Arizona State University.
1. Preparing Acid-cleaned Cover Glass
NOTE: Cover glass purchased from many manufacturers may not be as clean as expected. Consider routinely cleaning batches of cover glass before any procedure where microscopy is involved.
<ol>
	<li>Purchase high stringency cover glass. Take special care to choose the correct ...

<ol>
	<li>McNally, A. K., Anderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-4+induces+foreign+body+giant+cells+from+human+monocytes/macrophages.+Differential+lymphokine+regulation+of+macrophage+fusion+leads+to+morphological+variants+of+multinucleated+giant+cells.">Interleukin-4 induces foreign body giant cells from human monocytes/macrophages. Differential lymphokine regulation of macrophage fusion leads to morphological variants of multinucleated giant cells.</a> Am J Pathol. 147 (5), 1487 (1995).</li><li>Helming, L., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mediators+of+macrophage+fusion.">Molecular mediators of macrophage fusion.</a> Trends Cell Biol. 19 (10), 514-522 (2009).</li><li>Podolnikova, N. P., Kushchayeva, Y. S., Wu, Y., Faust, J., Ugarova, T. 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The need to identify and subsequently develop optical-quality glass surfaces that promote macrophage fusion stemmed from the fact that until recently no published study directly visualized macrophage fusion in the context of living specimens3,4. This is due to the fact that fusogenic plastic surfaces that are commonly used require LWD objectives and are largely limited to phase contrast optics. These barriers were overcome by engineering an optical-quality glass ...

The formation of MGCs accompanies a number of pathological states in the human body distinguished by chronic inflammation1. Despite agreement that mononucleated macrophages fuse to form MGCs2, surprisingly few studies have shown fusion in context with living specimens3,4. This is because clean glass surfaces that are required for most imaging techniques do not promote macrophage fusion when induced by inflammatory cytokines5. Indeed, if clean glass is used as a substrate for macrophage fusion, then low to intermediate magnification objectives (i.e., 10 - 20X) and more than 15 h of continuous imaging are often required to observe a single fusion event.
On the other hand, fusogenic plastic surfaces (e.g., permanox) or bacteriological grade plastic promote fusion2. However, imaging through plastic is problematic because the substrate is thick and scatters light. This complicates imaging since long working distance (LWD) objectives are required. However, LWD objectives usually have low light gathering capacity compared to their coverslip-corrected counterpart. Further, techniques that exploit changes in the polarity of light passing through the specimen such as differential interference contrast are impossible since plastic is birefringent. The barriers associated with the use of plastic are further underscored by the fact that it is impossible to predict where macrophage fusion/MGC formation will occur on the surface. Together, these limitations restrict the visualization of macrophage fusion to phase contrast optics, extended total imaging durations (&#62;15 continuous hours), and low resolution.
We recently identified a highly fusogenic glass surface while conducting single-molecule super resolution microscopy with fixed macrophages/MGCs4. This observation was surprising because clean glass surfaces promote fusion at the very low rate of ~ 5% after 24 h in the presence of interleukin-4 (IL-4) as determined by the fusion index4. We found that the capacity to promote fusion was due to oleamide contamination. Adsorption of oleamide or other compounds that similarly contained long-chain hydrocarbons made the glass fusogenic. The most fusogenic compound (paraffin wax) was micropatterned, and it imparted a high degree of spatiotemporal control over macrophage fusion and a 2-fold increase in the number of fusion events observed within the same amount of time compared to permanox. These optical-quality surfaces provided the first glimpse into the morphological features and kinetics that govern the formation of MGCs in living specimens.
In this protocol we describe the fabrication of a variety of glass surfaces that can be used to monitor the formation of MGCs from living specimens. In addition, we show that these surfaces are amenable to far-field super-resolution techniques. Surface fabrication is dependent on the goal of the experiment, and each surface is described with related examples in the proceeding text.

<table border="0" cellpadding="0" cellspacing="0" width="1000">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Plasma cleaner</td>
			<td style="width:240px;">Harrick Plasma</td>
			<td style="width:129px;">PCD-32G</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Finder grid</td>
			<td>Electron microscopy sciences</td>
			<td>G400F1-Au</td>
			<td>any gold TEM grid will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass (22x22 mm)</td>
			<td>Thor Labs</td>
			<td>CG15CH</td>
			<td>use only high stringency cover glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraffin wax</td>
			<td>Sigma Aldrich</td>
			<td>17310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petrolatum</td>
			<td>Sigma Aldrich</td>
			<td>16415</td>
			<td>must be &#945;-tocopherol-free if substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oleamide</td>
			<td>Sigma Aldrich</td>
			<td>O2136</td>
			<td>prepare fresh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td>Sigma Aldrich</td>
			<td>278475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toluene</td>
			<td>Sigma Aldrich</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td>VWR International</td>
			<td>BDH1101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Electron microscopy sciences</td>
			<td>15050</td>
			<td>use low dissolved solids ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Fischer Scientific</td>
			<td>A144C-212</td>
			<td>use to acid wash cover glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slyguard 184</td>
			<td>VWR International</td>
			<td>102092-312</td>
			<td>mix in a 1:10 ratio and cure at 50 &#176;C for 4 h</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm petri dish</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-351864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont no. 5 forceps</td>
			<td>Electron microscopy sciences</td>
			<td>72705</td>
			<td>ideal for removing TEM grid in section 3.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Atlanta Biological</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM:F12</td>
			<td>Corning</td>
			<td>10-092</td>
			<td>contains 15 mM HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strept</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS</td>
			<td>Corning</td>
			<td>21-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA solution</td>
			<td>Sigma Aldrich</td>
			<td>A9576</td>
			<td>use at 0.1% in HBSS to wash non-adherent macrophages</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IL-4</td>
			<td>Genscript</td>
			<td>Z02996</td>
			<td>aliquot at 10 &#956;g/mL and store at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6J</td>
			<td>Jackson Laboratory</td>
			<td>000664</td>
			<td>use for fixed samples or techniques that do not require contrast agents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eGFP-LifeAct mice</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>use for live fluorescence imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipe</td>
			<td>Kimberly Clark&#160;</td>
			<td>34155</td>
			<td>use to polish hydrocarbon adsorbed surfaces</td>
		</tr>
	</tbody>
</table>

fabricating optical-quality glass surfaces to study macrophage fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

Physicochemical parameters of materials have dramatic effects on the extent of macrophage fusion7,8,9,10. Moreover, surface contaminants are known to promote macrophage fusion11. Therefore, it is important to start with clean cover glass as a negative control for macrophage fusion. When cleaned as described in protocol 1, the cover glass ...

Watch this Scientific Journal Video about Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion at JoVE.com

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

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