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

我们要感谢 Ugarova 实验室的成员和代谢和血管生物学中心的调查人员, 以便对这项工作进行有益的讨论。詹姆斯浮士德希望在2015年对欧洲分子生物学实验室超分辨率显微镜课程的导师表示感谢。我们感谢萨特雅·南丹 Khuon 在 Janelia 的帮助, 为 LLSM 做样品准备。在这项工作的审查和摄制部分詹姆斯浮士德由 T32 奖学金 (5T32DK007569-28) 支持。这项工作的晶格光片组件由 HHMI 和贝蒂和戈登摩尔基金会支持。歌迷由 NIH 赠款 HL63199 资助。

使用动物的程序是在梅奥诊所、Janelia 研究校区和亚利桑那州立大学的动物护理和使用委员会批准的。
1. 制备酸性清洗罩玻璃
注: 从许多制造商购买的盖玻璃可能不像预期的那样干净。在任何涉及显微术的程序之前, 请考虑例行清洗覆盖玻璃批次。
<ol><li>采购高紧缩盖玻璃。请特别注意选择正确的厚度 (0.15 或0.17 毫米)。 注: 覆盖玻璃厚度的选择取决于...

<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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Integrins+&#945;+M+&#946;+2+(Mac-1,+CD11b/CD18)+and+&#945;+D+&#946;+2+(CD11d/CD18)+in+Macrophage+Fusion.">The Role of Integrins &#945; M &#946; 2 (Mac-1, CD11b/CD18) and &#945; D &#946; 2 (CD11d/CD18) in Macrophage Fusion.</a> Am J Pathol. 186 (8), 2105-2116 (2016).</li><li>Faust, J. J., Christenson, W., Doudrick, K., Ros, R., Ugarova, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+fusogenic+glass+surfaces+that+impart+spatiotemporal+control+over+macrophage+fusion:+Direct+visualization+of+multinucleated+giant+cell+formation.">Development of fusogenic glass surfaces that impart spatiotemporal control over macrophage fusion: Direct visualization of multinucleated giant cell formation.</a> Biomaterials. 128, 160-171 (2017).</li><li>Jenney, C. R., 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=Alkylsilane-modified+surfaces:+Inhibition+of+human+macrophage+adhesion+and+foreign+body+giant+cell+formation.">Alkylsilane-modified surfaces: Inhibition of human macrophage adhesion and foreign body giant cell formation.</a> J Biomed Mater Res. 46 (1), 11-21 (1999).</li><li>Vignery, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+fuse+macrophages+in+vitro.">Methods to fuse macrophages in vitro.</a> Cell Fusion: Overviews Methods. , 383-395 (2008).</li><li>Zhao, Q., 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=Foreign-body+giant+cells+and+polyurethane+biostability:+In+vivo+correlation+of+cell+adhesion+and+surface+cracking.">Foreign-body giant cells and polyurethane biostability: In vivo correlation of cell adhesion and surface cracking.</a> J Biomed Mater Res. 25 (2), 177-183 (1991).</li><li>Anderson, J. M., Rodriguez, A., Chang, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foreign+body+reaction+to+biomaterials.">Foreign body reaction to biomaterials.</a> Semin Immunol. 20 (2), 86-100 (2008).</li><li>Jenney, C. R., 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=Effects+of+surface-coupled+polyethylene+oxide+on+human+macrophage+adhesion+and+foreign+body+giant+cell+formation+in+vitro.">Effects of surface-coupled polyethylene oxide on human macrophage adhesion and foreign body giant cell formation in vitro.</a> J Biomed Mater Res. 44 (2), 206-216 (1999).</li><li>DeFife, K. M., Colton, E., Nakayama, Y., Matsuda, T., 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=Spatial+regulation+and+surface+chemistry+control+of+monocyte/macrophage+adhesion+and+foreign+body+giant+cell+formation+by+photochemically+micropatterned+surfaces.">Spatial regulation and surface chemistry control of monocyte/macrophage adhesion and foreign body giant cell formation by photochemically micropatterned surfaces.</a> J Biomed Mater Res. 45 (2), 148-154 (1999).</li><li>Jenney, C. R., DeFife, K. M., Colton, E., 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=Human+monocyte/macrophage+adhesion,+macrophage+motility,+and+IL-4-induced+foreign+body+giant+cell+formation+on+silane-modified+surfaces+in+vitro.">Human monocyte/macrophage adhesion, macrophage motility, and IL-4-induced foreign body giant cell formation on silane-modified surfaces in vitro.</a> J Biomed Mater Res. 41 (2), 171-184 (1998).</li><li>Hell, S. W., Wichmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+diffraction+resolution+limit+by+stimulated+emission:+stimulated-emission-depletion+fluorescence+microscopy.">Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.</a> Opt Lett. 19 (11), 780-782 (1994).</li><li>van de Linde, 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=Direct+stochastic+optical+reconstruction+microscopy+with+standard+fluorescent+probes.">Direct stochastic optical reconstruction microscopy with standard fluorescent probes.</a> Nat Protocols. 6 (7), 991-1009 (2011).</li><li>F&#246;lling, 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=Fluorescence+nanoscopy+by+ground-state+depletion+and+single-molecule+return.">Fluorescence nanoscopy by ground-state depletion and single-molecule return.</a> Nat Methods. 5 (11), 943-945 (2008).</li><li>Heilemann, 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=Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes.">Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.</a> Ange Chem Int Edit. 47 (33), 6172-6176 (2008).</li><li>Betzig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proposed+method+for+molecular+optical+imaging.">Proposed method for molecular optical imaging.</a> Optics Letters. 20 (3), 237-239 (1995).</li><li>Betzig, E., 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=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Shtengel, 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=Interferometric+fluorescent+super-resolution+microscopy+resolves+3D+cellular+ultrastructure.">Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.</a> P Natl Acad Sci U S A. 106 (9), 3125-3130 (2009).</li><li>Shtengel, 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=Imaging+cellular+ultrastructure+by+PALM,+iPALM,+and+correlative+iPALM-EM.">Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.</a> Method Cell Biol. 123, 273-294 (2013).</li><li>Chen, B. 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=Lattice+light-sheet+microscopy:+Imaging+molecules+to+embryos+at+high+spatiotemporal+resolution.">Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution.</a> Science. 346 (6208), 1257998 (2014).</li><li>Planchon, T. 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=Rapid+three-dimensional+isotropic+imaging+of+living+cells+using+Bessel+beam+plane+illumination.">Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination.</a> Nat Methods. 8 (5), 417-423 (2011).</li></ol>

需要识别并随后开发出能促进巨噬细胞融合的光学质量玻璃表面, 这是因为直到最近没有发表的研究在活体标本的背景下直接可视化巨噬细胞融合3, 4。这是因为, 通常使用的融合塑料表面需要 LWD 目标, 并且主要限于相位对比光学。这些障碍是通过工程的光学质量玻璃表面, 不仅促进极高的巨噬细胞融合率, 但传授了惊人的空间控制 MGCs 的形成。
<p class...

MGCs 的形成伴随着人体的一些病理状态, 由慢性炎症1来区分。尽管同意 mononucleated 巨噬细胞保险丝形成 MGCs2, 惊奇地很少研究显示了融合在上下文与活体标本3,4。这是因为大多数成像技术所需的干净玻璃表面在炎症细胞因子5诱导下不会促进巨噬细胞融合。事实上, 如果用干净的玻璃作为巨噬细胞融合的基底, 那么低到中等放大目标 (即, 10-20X) 和超过 15 h 的连续成像通常需要观察一个单一的融合事件。
另一方面, 融合塑料表面 (例如, permanox) 或细菌级塑料促进融合2。然而, 通过塑料成像是有问题的, 因为基板是厚和散射光。这使得成像变得复杂, 因为需要长的工作距离 (LWD) 目标。然而, 与盖玻片校正的相对物相比, LWD 目标通常具有较低的光收集能力。此外, 由于塑料是双折射的, 利用通过试样如差动干涉对比的光线极性变化的技术是不可能的。与使用塑料有关的障碍进一步被强调的事实是, 它是不可能预测巨噬细胞融合/MGC 形成将发生在表面上。这些限制共同限制了巨噬细胞融合对相对比光学的可视化, 延长的总成像持续时间 (> 15 小时) 和低分辨率。
我们最近发现了一个高度融合的玻璃表面, 同时进行单分子超分辨率显微镜与固定巨噬细胞/MGCs4。这种观察是令人惊讶的, 因为干净的玻璃表面促进融合在极低率的5% 后24小时的存在 interleukin-4 (IL-4), 由融合指数4确定。我们发现促进聚变的能力是由于 oleamide 污染造成的。吸附 oleamide 或其他类似于长链碳氢化合物的化合物使玻璃融合。最融合化合物 (石蜡) 是 micropatterned, 它传授了高度的时空控制巨噬细胞融合和2倍增加的融合事件的数量在相同的时间内观察到与 permanox 相比。这些光学质量的表面提供了第一次一瞥的形态特征和动力学, 控制 MGCs 的形成在活体标本。
在本议定书中, 我们描述了各种玻璃表面的制造, 可用于监测从活体标本中 MGCs 的形成。此外, 我们还表明, 这些表面是适于远场超分辨率技术。表面加工依赖于实验的目标, 每个表面都用相关的例子来描述。

<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

从活体标本中多核巨细胞 (MGCs) 的形成可视化是一个挑战, 因为大多数活的成像技术需要通过玻璃传播光, 但在玻璃巨噬细胞融合是一个罕见的事件。该协议提供了几个光学质量玻璃表面的制造, 在这些材料中, 吸附含有长链碳氢化合物的化合物将玻璃转化为融合表面。首先, 介绍了清洁玻璃表面作为表面改性起始材料的制备方法。其次, 提出了一种吸附长链烃类化合物的方法, 将非融合玻璃转化为融合基体。第三, 该协议描述了表面 micropatterns 的制造, 促进了对 MGC 形成的高度时空控制。最后, 介绍了玻璃底菜的制作。用此体外细胞系统作为研究巨噬细胞融合和 MGC 形成模型的例子。

材料的物理化学参数对巨噬细胞融合的程度有戏剧性的影响7,8,9,10。此外, 已知的表面污染物促进巨噬细胞融合11。因此, 对巨噬细胞融合进行负控制, 首先要从清洁罩玻璃开始。如1号协议所述, 盖玻璃是异常平坦的, 没有明显的表面特征, 而长链碳氢化合物的?...

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

该协议描述了光学质量玻璃表面的制备, 这些化合物含有长链碳氢化合物, 可用于监测活体标本的巨噬细胞融合, 并能实现固定标本的超分辨显微术。.

制备光学质量玻璃表面研究巨噬细胞融合

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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Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

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

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

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

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

利用共焦显微镜可视化巨噬细胞胞外陷阱

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

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

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

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