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 ...

The overall goal of this protocol is to transform non-fusogenic cover glass into a fusogenic surface that can be used to study macrophage fusion using any form of optical microscopy. This method can help to answer the fundamental questions related to the molecular mechanism of macrophage fusion. The main advantage of this procedure is that any microscope technique, particularly live imaging, can be used to study macrophage fusion.Demonstrating the procedure will be James Faust, third fellow, and Arnat Balabiyev, a graduate student from my laboratory. To begin, in a well-ventilated chemical fume hood incubate high-stringency cover glass in 12-molar hydrochloric acid with sonication for one hour. Repeat the step two additional times with fresh 12-molar HCL.Fill a separate beaker with ultrapure water, and add the cover glass. Then sonicate the cover glass for five to 10 minutes. Next, incubate the cover glass in pure acetone for 30 minutes with sonication.Repeat the step two additional times. Then fill a separate beaker with sterile ultrapure water, and add the cover glass. Sonicate the cover glass for five to 10 minutes.Incubate the cover glass in 100%ethyl alcohol for 30 minutes with sonication. Repeat this step two additional times. For longterm storage, place the acid-cleaned cover glass in a container willed with pure ethyl alcohol.Alternatively, dry the cover glass with nitrogen gas, and store in a vacuum desiccator. Dissolve DMSO-free high-melting-point paraffin wax in ultrapure toluene. Using a glass pipette, apply enough paraffin working solution to a dry acid-cleaned cover glass to evenly coat the glass surface.Decant the excess solution, and with nitrogen gas or air, dry the cover glass. With a lint-free wipe, polish the cover glass using three strokes along the X axis, then three strokes along the Y axis. Immediately before the experiment is conducted, use sterile ultrapure water to wash the cover glass, and subsequently sterilize it with ultraviolet light in a biological safety hood for 15 to 30 minutes.Dry the acid-cleaned cover glass, and immobilize the glass on a flat surface. Using forceps, carefully immerse a gold transmission electron microscopy finder grid in a working solution of a hydrocarbon compound. Wick away excess solution by gently tapping the grid on filter paper, then immediately place the grid in the center of the cover glass.Allow the toluene to dry for two minutes. Make sure the grid is bonded to the glass by gently inverting the glass. If the grid detaches, repeat the bonding using a new acid-washed cover glass.After removing the TEM grid, examine the continuity of the micro pattern with low magnification. The micro pattern should appear continuous, but the material should not extend beyond the margins of the pattern. Next, use a plasma cleaner with vacuum-gas plasma to clean the cover glass according to the text protocol.Then, using fine-tip forceps, carefully remove the grid from the glass surface to expose the micro pattern. With a step drill bit, drill a six to 10 millimeter circular hole with smooth edges in the bottom of a 35-millimeter plastic petri dish. Carefully mix and degas silicon elastomer according to the manufacturer's instructions.Apply a thin coating of elastomer just proximate to the edge of the hole in the petri dish. The elastomer should appear as a continuous thin band surrounding the hole. Gently apply either a fusogenic or the dry acid-cleaned cover glass to the dish in order to cover the hole surrounded by elastomer.Ensure that the cover glass appears flush with the bottom of the dish and extends beyond the diameter of the hole so that a substantial portion of the glass is in contact with the plastic. Cure the elastomer by baking it at 50 degrees Celsius for two to three hours. Inject eight-week-old C57 black-6 mice with 0.5 milliliters of a sterile solution of 4%Brewer's Thioglycolate as outlined in the text protocol.72 hours later, after euthanizing the animal according to approved animal care and use guidelines, collect macrophage by interperitoneal lavage using ice-cold PBS supplemented with five-millimolar EDTA. Centrifuge the macrophages at 300 times G for three minutes, and resuspend the cell pellet in one milliliter of DMEM/F-12 supplemented with 15 millimolar heaps 10%FBS and 1%antibiotics. After counting and diluting the cells and applying them to the surfaces for 30 minutes, use HBSS 0.1%BSA to wash the surfaces, and replace the culture medium.Then return the cultures to the incubator. Two hours later, aspirate the medium, and replace it with culture medium supplemented with 10 nanograms per milliliter of IL4 before imaging the cells. When cleaned as described in this video, the cover glass is exceptionally flat with no obvious surface features;whereas the absorption of long-chain hydrocarbons followed by surface polishing creates a degree of nanotopography seen here.On clean cover-glass surfaces macrophages fuse at a very low rate. In contrast, after 24 hours in the presence of IL4, the macrophages applied to surfaces adsorbed with compounds containing long-chain hydrocarbons undergo robust fusion. Further, absorption of solvent alone does not make glass a fusogenic surface.Here, full width at half maximum, or FWHM, of structures smaller than the diffraction limit of the light microscope is used to quantitatively assess the potential differences in resolution. Importantly, there is no apparent difference in FWHM of 100-nanometer fluorescent beads among the various surfaces, suggesting that the characteristics of glass required for fluorescence imaging were sufficiently preserved. In this experiment macrophages were applied to the surfaces containing long-chain hydrocarbons.Then a form of super-resolution microscopy called 3D Direct Stochastic Optical Reconstruction Microscopy was carried out. These videos demonstrate that a variety of live-imaging techniques are feasible when macrophages are applied to the surfaces. Once mastered, modifying the glass to make the surface fusogenic can be done in half an hour if it is properly done.While attempting this procedure it is important to remember to follow it carefully. Following this procedure, other methods, like fluorescence microscopy, can be performed in order to obtain views of fusion with molecule-specific contrast. This will enable experiments designed to identify proteins directly involved at the time of fusion.After its development, this technique paved the way for investigators to acquire time-resolved views of macrophage fusion. In the future, this will help us to better understand the mechanism of macrophage fusion. After watching this video, you should be able to modify cover glass to study macrophage fusion.Please don't forget that working with acid and toluene can be extremely hazardous, and PPE and proper ventilation should always be used while performing this procedure.

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A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...