Name: mesenchymal stem cell regulation of macrophage phagocytosis; quantitation and imaging
Uploaded: 2021-07-16T14:00:00
Description: Watch this Scientific Journal Video about Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging at JoVE.com

这项工作得到了NSF主要研究仪器机制的支持，其授权号为1626093和1919583。

注意：所有培养基制备和细胞培养技术均在无菌条件下使用具有层流流动的生物安全柜进行。所描述的所有培养步骤均使用旨在保持37°C，5%CO2和95%湿度的培养箱进行。
1. 细胞培养
<ol><li>生长培养基的制备<ol>
<li>对于 MSC 和 LADMAC，将 50 mL FBS 和 5 mL 100x 抗生素/抗真菌剂混合物加入 500 mL 高糖 DMEM 中。</li>
		<li>对于MΦ，加入50 mL FBS，100 mL LADMAC条件培养基（按照第1.2节的?...

<ol>
	<li>Phinney, D. G., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Mesenchymal+stem/multipotent+stromal+cells:+The+state+of+transdifferentiation+and+modes+of+tissue+repair--current+views.">Concise review: Mesenchymal stem/multipotent stromal cells: The state of transdifferentiation and modes of tissue repair--current views.</a> Stem Cells. 25 (11), 2896-2902 (2007).</li><li>Bernardo, M. E., Fibbe, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+sensors+and+switchers+of+inflammation.">Mesenchymal stromal cells: sensors and switchers of inflammation.</a> Cell Stem Cell. 13 (4), 392-402 (2013).</li><li>Tobin, L. M., Healy, M. E., English, K., Mahon, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+suppress+donor+CD4(+)+T+cell+proliferation+and+reduce+pathology+in+a+humanized+mouse+model+of+acute+graft-versus-host+disease.">Human mesenchymal stem cells suppress donor CD4(+) T cell proliferation and reduce pathology in a humanized mouse model of acute graft-versus-host disease.</a> Clinical and Experimental Immunology. 172 (2), 333-348 (2013).</li><li>Giuliani, 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=Long-lasting+inhibitory+effects+of+fetal+liver+mesenchymal+stem+cells+on+T-lymphocyte+proliferation.">Long-lasting inhibitory effects of fetal liver mesenchymal stem cells on T-lymphocyte proliferation.</a> PLoS One. 6 (5), 19988 (2011).</li><li>Plumas, J., Chaperot, L., Richard, M. J., Molens, J. P., Bensa, J. C., Favrot, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+induce+apoptosis+of+activated+T+cells.">Mesenchymal stem cells induce apoptosis of activated T cells.</a> Leukemia. 19 (9), 1597-1604 (2005).</li><li>Luz-Crawford, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+generate+a+CD4+CD25+Foxp3++regulatory+T+cell+population+during+the+differentiation+process+of+Th1+and+Th17+cells.">Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells.</a> Stem Cell Research &amp; Therapy. 4 (3), 65 (2013).</li><li>Waterman, R. S., Tomchuck, S. L., Henkle, S. L., Betancourt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mesenchymal+stem+cell+(MSC)+paradigm:+Polarization+into+a+pro-inflammatory+MSC1+or+an+Immunosuppressive+MSC2+phenotype.">A new mesenchymal stem cell (MSC) paradigm: Polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype.</a> PLoS One. 5 (4), 10088 (2010).</li><li>Nemeth, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow+stromal+cells+attenuate+sepsis+via+prostaglandin+E(2)-dependent+reprogramming+of+host+macrophages+to+increase+their+Interleukin-10+production.">marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their Interleukin-10 production.</a> Nature Medicine. 15 (1), 42-49 (2009).</li><li>Maggini, 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=Mouse+Bone+Marrow-Derived+Mesenchymal+Stromal+Cells+Turn+Activated+Macrophages+into+a+Regulatory-Like+Profile.">Mouse Bone Marrow-Derived Mesenchymal Stromal Cells Turn Activated Macrophages into a Regulatory-Like Profile.</a> PLoS One. 5 (2), 9252 (2010).</li><li>Takizawa, N., 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=marrow-derived+mesenchymal+stem+cells+propagate+immunosuppressive/anti-inflammatory+macrophages+in+cell-to-cell+contact-independent+and-dependent+manners+under+hypoxic+culture.">marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and-dependent manners under hypoxic culture.</a> Experimental Cell Research. 358 (2), 411-420 (2017).</li><li>Kim, J., Hematti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-educated+macrophages:+a+novel+type+of+alternatively+activated+macrophages.">Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages.</a> Experimental Hematology. 37 (12), 1445-1453 (2009).</li><li>Evans, J. F., Salvador, V., George, S., Trevino-Gutierrez, C., Nunez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+aorta-derived+mesenchymal+progenitor+cells+contribute+to+and+enhance+the+immune+response+of+macrophage+cells+under+inflammatory+conditions.">Mouse aorta-derived mesenchymal progenitor cells contribute to and enhance the immune response of macrophage cells under inflammatory conditions.</a> Stem Cell Research &amp; Therapy. 6 (1), 56 (2015).</li><li>Cho, D. I., 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=Mesenchymal+stem+cells+reciprocally+regulate+the+M1/M2+balance+in+mouse+bone+marrow-derived+macrophages.">Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages.</a> Experimental Molecular Medicine. 46, 70 (2014).</li><li>Fernandez, N., 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=Mouse+mesenchymal+progenitor+cells+expressing+adipogenic+and+osteogenic+transcription+factors+suppress+the+macrophage+inflammatory+response.">Mouse mesenchymal progenitor cells expressing adipogenic and osteogenic transcription factors suppress the macrophage inflammatory response.</a> Stem Cells International. 2017, 5846257 (2017).</li><li>Jackson, M. V., 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=Mitochondrial+transfer+via+tunneling+nanotubes+is+an+important+mechanism+by+which+mesenchymal+stem+cells+enhance+macrophage+phagocytosis+in+the+in+vitro+and+in+vivo+models+of+ARDS.">Mitochondrial transfer via tunneling nanotubes is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ARDS.</a> Stem Cells. 34 (8), 2210-2223 (2016).</li><li>Chaplin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+immune+response.">Overview of the immune response.</a> The Journal of Allergy and Clinical Immunology. 125 (2), 3-23 (2010).</li><li>Lim, 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=Characterizing+the+mechanisms+of+nonopsonic+uptake+of+cryptococci+by+macrophages.">Characterizing the mechanisms of nonopsonic uptake of cryptococci by macrophages.</a> Journal of Immunology. 200 (10), 3539-3546 (2018).</li><li>Wang, Z., 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=Interferon-&#947;+inhibits+nonopsonized+phagocytosis+of+macrophages+via+an+mTORC1-c/EBP+pathway.">Interferon-&#947; inhibits nonopsonized phagocytosis of macrophages via an mTORC1-c/EBP pathway.</a> Journal of Innate Immunity. 7 (2), 165-176 (2015).</li><li>Lindner, B., Burkard, T., Schuler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+assays+with+different+PH-sensitive+fluorescent+particles+and+various+readouts.">Phagocytosis assays with different PH-sensitive fluorescent particles and various readouts.</a> Biotechniques. 68, 245-250 (2020).</li><li>Kapellos, T. 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=A+novel+real+time+imaging+platform+to+quantify+macrophage+pahgocytosis.">A novel real time imaging platform to quantify macrophage pahgocytosis.</a> Biochemical Pharmacology. 116, 107-119 (2016).</li><li>Takahashi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+quantitation+of+platelet+phagocytosis+by+monocytes+using+a+pH-sensitive+dye,+pHrodo-SE.">Flow cytometric quantitation of platelet phagocytosis by monocytes using a pH-sensitive dye, pHrodo-SE.</a> Journal of Immunological Methods. 447, 57-64 (2017).</li></ol>

使用与pH敏感染料偶联的生物颗粒分析吞噬作用是一种相对较新的工具，已被证明比传统的荧光标记颗粒12，19，20具有优势。对于传统的荧光标记颗粒，只有终点分析是可行的。在洗涤或淬灭未被吞噬细胞吸收的颗粒后，使用荧光显微镜和/或光谱荧光测定法进行检测。从光谱荧光测定法获得的定量数据具有检测非吞噬颗粒的潜...

间充质干细胞（MSC）是产生结缔组织细胞的祖细胞。MSC存在于成年哺乳动物组织中，可以从骨髓中分离出来1。由于其免疫调节特性，这些细胞被广泛研究2。早期的研究集中在MSC调节T细胞3，4，5，6，但最近，它们对巨噬细胞（MΦ）的调节，巨噬细胞是先天免疫的主要细胞成分，已经受到越来越多的关注7，8，9，10，11，12，13，14.MSC-MΦ相互作用在炎症性疾病治疗中的重要性通过单核细胞/巨噬细胞的消耗在动物模型8中废除MSC的治疗效果这一事实得到了强调。这里的重点是MSC与MΦ的细胞接触相互作用。MSC有能力通过促进从炎症反应到抗炎反应的转变来调节MΦ的表型，导致组织修复活动8，9，10，11，并且已经做了很多工作来证明这些调节机制。在其他情况下，MSC可以支持或加剧MΦ驱动的炎症反应12，13并增强MΦ吞噬活性14，15。然而，严重缺乏现有数据来确定MSC调节MΦ吞噬活性的机制和条件。
MΦ具有识别调理子化（抗体或补体包被）或非调序化病原体导致吞噬作用的受体家族16。后者的激活和活性研究较少17.在非炎症性体外环境中，MSC增强非调子化病原体的MΦ吞噬作用13。然而，在适应性免疫应答期间暴露于淋巴细胞产生的炎症环境后，MΦ对非调序化病原体的识别可能会减少。由自然杀伤细胞和效应淋巴细胞释放的IFN-γ对非调子化颗粒18的MΦ吞噬作用具有抑制作用。建立了共培养模型，研究了MSC直接接触调节MΦ吞噬作用的机制。这里提出的实验目标是确定在MΦ暴露于IFN-γ后，MSC是否调节非调理性病原体的MΦ吞噬作用（图1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 Well Black Polystyrene Microplate</td>
			<td>MilliporeSigma</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.4% trypan blue solution</td>
			<td>MilliporeSigma</td>
			<td>T8154-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL and 50 mL Conical Sterile Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well Chambered Coverglass w/ non-removable wells</td>
			<td>ThermoFisher</td>
			<td>155382PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)&#160;Gibco</td>
			<td>ThermoFisher</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Axiobserver 7 Imaging System</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>MilliporeSigma</td>
			<td>A8806-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>MilliporeSigma</td>
			<td>CLS3008-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks, tissue culture treated, surface area 75&#160;cm2, canted neck, with cap, filtered</td>
			<td>MilliporeSigma</td>
			<td>C7231-120EA</td>
			<td></td>
		</tr>
		<tr>
			<td>D1 ORL UVA [D1]</td>
			<td>ATCC</td>
			<td>CRL-12424</td>
			<td>Mouse MSC Cell Line</td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>ThermoFisher</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>FisherScientific</td>
			<td>02-671-51B</td>
			<td></td>
		</tr>
		<tr>
			<td>I-11.15</td>
			<td>ATCC</td>
			<td>CRL-2470</td>
			<td>Mouse M&#934; Cell Line</td>
		</tr>
		<tr>
			<td>LADMAC Cell Line</td>
			<td>ATCC</td>
			<td>CRL-2420</td>
			<td>LADMAC cells secrete the growth factor colony stimulating factor 1 (CSF-1).</td>
		</tr>
		<tr>
			<td>Live-Cell Imaging solution</td>
			<td>ThermoFisher</td>
			<td>A14291DJ</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>ThermoFisher</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Green Zymosan Bioparticles Conjugate</td>
			<td>ThermoFisher</td>
			<td>P35365</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IFN-&#947;</td>
			<td>Preprotech</td>
			<td>315-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectramax i3X</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Single Use Vacuum Filter Units, 250 mL, 0.2 &#181;m</td>
			<td>ThermoFisher</td>
			<td>568-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe filters, 0.2 micrometer</td>
			<td>ThermoFisher</td>
			<td>723-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture treated culture dishes, 100 mm x 20 mm</td>
			<td>MilliporeSigma</td>
			<td>CLS430167-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>ThermoFisher</td>
			<td>25300054</td>
			<td></td>
		</tr>
	</tbody>
</table>

mesenchymal stem cell regulation of macrophage phagocytosis; quantitation and imaging

间充质干细胞（MSC）传统上因其再生特性而受到研究，但最近，它们的免疫调节特性一直处于最前沿。它们与免疫细胞相互作用并调节免疫细胞活性。本研究的重点是巨噬细胞吞噬活性的MSC调节。巨噬细胞（MΦ）吞噬作用是先天免疫系统对感染反应的重要组成部分，MSC调节这种反应的机制正在积极研究中。这里介绍的是一种研究与MSC共培养时与pH敏感荧光分子偶联的非调序酶原颗粒的MΦ吞噬作用的方法。随着吞噬活性的增加和标记的酶聚糖颗粒被封闭在吞噬体的酸性环境中，pH敏感分子的荧光强度增加。在适当的激发和发射波长下，使用荧光分光光度计测量吞噬活性，并将动力学数据显示为70分钟内相对荧光单位的变化。为了支持这些定量数据，使用动态成像可视化吞噬细胞活性的变化。使用该方法的结果表明，在共培养中，MSC增强了幼稚和IFN-γ处理的MΦ的非调子化酶的MΦ吞噬作用。这些数据增加了目前对MSC调节先天免疫系统的知识。这种方法可以应用于未来的研究，以充分描绘潜在的细胞和分子机制。

在所有时间点计算每组的平均±SEM后，数据以折线图格式显示，Y轴作为相对荧光强度，X轴作为时间。 补充文件1 以电子表格格式提供了来自96孔板动力学读取的原始数据的示例。
在这项研究中，图3A和表3中呈现的最佳结果表明，1）与MSC共培养增强巨噬细胞的吞噬活性，2）IFN-y处理降低巨噬细胞的活性，以及3）与MSC共培养部分?...

Watch this Scientific Journal Video about Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging at JoVE.com

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

这里提出的方案用于量化和生成间充质干细胞（MSC）介导的巨噬细胞（MΦ）吞噬作用的动态图像，这些聚氧菌（zymosan）颗粒与pH敏感荧光分子偶联。

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

这种共培养方法将有助于回答围绕间充质干细胞调节巨噬细胞吞噬作用背后的细胞接触机制的关键问题，从而阐明MSC在调节先天免疫中的作用。此技术可应用于高通量分析。与pH敏感染料偶联的生物颗粒仅在酸性吞噬体环境中发出荧光。因此，清洗和淬火是必要的。该方法还可以应用于包括嗜中性粒细胞和其他吞噬细胞在内的各种共培养模型。演示该程序的将是Ethan Chetkof，他是目前在我实验室的本科生。首先，观察培养的间充质干细胞的汇合。当达到70-80%汇合度时，在100毫米培养皿中从培养的细胞中吸出生长培养基并加入5毫升PBS。吸出PBS后，加入两毫升0.05%胰蛋白酶EDTA溶液，孵育细胞三分钟。三分钟后，使用倒置显微镜检查细胞脱离。如果细胞没有分离，再次孵育一到两分钟。一旦所有细胞分离，向平板中加入五毫升新鲜生长培养基。使用胰蛋白酶培养基混合物冲洗板后，使用无菌血清学移液管将细胞收集到干净，无菌的50毫升锥形管中。接下来，通过离心将细胞沉淀下来。吸出上清液后，通过轻轻上下移液将细胞沉淀重悬于10毫升新鲜生长培养基中。对于细胞计数，将10微升细胞悬浮液加入含有30微升0.4%台盼蓝溶液的1.5毫升微量离心管中，然后在血细胞计数器计数室的盖玻片下加入10微升混合物。在倒置或明场显微镜下，使用10倍物镜和四个一平方毫米腔计算未染色的活细胞，并计算文本手稿中描述的细胞数。使用公式C1V1等于C2V2来确定制备最终浓度为每毫升10至第五个细胞的细胞悬浮液所需的新鲜培养基和细胞悬浮液的所需体积。根据板设计，使用多通道微量吹管在96孔板的孔中加入100微升的细胞悬浮液。使用微量移液管根据板设计向每个腔室载玻片中加入530微升的细胞悬浮液并孵育过夜。制备10毫升含有干扰素γ的活化培养基，浓度为每毫升250纳克，用于在一个100毫米培养皿中活化巨噬细胞。从巨噬细胞培养物中吸出生长培养基，并用不含干扰素γ的五毫升活化培养基冲洗细胞。在实验皿中吸入冲洗培养基后，加入10毫升补充有γ-干扰素的活化培养基。在对照皿中，加入10毫升不含干扰素γ的活化培养基，然后将细胞孵育16至24小时。在孵育结束时，为了制备共培养物，从巨噬细胞中除去活化培养基并加入5毫升新鲜生长培养基。使用细胞提升器轻轻刮擦细胞并将细胞收集到50毫升的锥形管中。接下来，使用台盼蓝排除和血液细胞术计数细胞，然后制备对照和处理过的巨噬细胞悬浮液，浓度为每毫升10至第五个细胞，如前所述用于间充质干细胞。从含有间充质干细胞的实验孔中轻轻吸出培养基。使用多通道微量移液器根据板设计在适当的实验孔中加入100微升处理的对照巨噬细胞悬浮液，并如图所示孵育过夜。从含有间充质干细胞的四壁腔载玻片中轻轻吸出培养基。使用微量移液器根据设计将530微升巨噬细胞悬浮液加入适当的孔中并孵育过夜。将一毫升活细胞成像培养基加入含有一毫克Zymosan颗粒的小瓶中，并将培养基颗粒混合物收集到玻璃培养管中。在用额外的一毫升成像溶液冲洗小瓶后，将冲洗过的成像介质转移到玻璃管中，然后加入三毫升额外的成像溶液，以达到每毫升0.2毫克的Zymosan颗粒悬浮液。使用快速脉冲涡旋Zymosan悬浮液30至60秒，然后使用探针超声仪用60个快速脉冲对悬浮液进行超声处理。吸出培养基后，用100微升活细胞成像溶液冲洗实验孔和试剂空白孔。接下来，从孔中吸出活细胞成像溶液，并加入100微升的Zymosan悬浮液。使用触摸板界面打开荧光读取器的板托盘。将不带盖子的板放在托盘中，A1孔位于左上角。使用触摸板关闭托盘，然后单击顶部菜单中的绿色读取按钮。为了进行吞噬作用测定，在用750微升成像培养基冲洗第一个实验井后，加入400微升的Zymosan悬浮液，将剩余的孔留在生长培养基中。接下来，将载玻片放在成像系统的培养台上。使用映像软件。选择定位选项卡下的明场选项，然后单击目镜图标。10倍物镜就位后，使用目镜和对焦旋钮聚焦在细胞上。选择采集选项卡。打开实验下拉菜单，选择一组波长，其中包括EGFP滤光片，以适应pH敏感染料的509纳米激发和533纳米发射波长。当计时器还剩下大约两分钟时，单击物镜菜单中的20X图标将物镜更改为20X，然后单击通道菜单，选择EGFP滤镜，然后在曝光菜单中，将曝光时间设置为400毫秒以检测pH敏感的绿色荧光团，然后单击实时。调整焦点后，单击停止以关闭灯光，然后选中顶部延时实验设置旁边的框。在对焦策略菜单中，选择软件自动对焦，然后从下拉菜单中选择智能和核心设置，以减少在自动对焦运行时暴露在光线下的时间。在延时菜单中，将所需的采集次数设置为30到60，间隔时间设置为一分钟，然后单击开始实验按钮开始采集。研究了干扰素γ对共培养物的影响。代表性结果表明，巨噬细胞与间充质干细胞的共培养增强了巨噬细胞的吞噬活性。干扰素γ处理降低巨噬细胞的活性。然而，在间充质干细胞存在下，巨噬细胞吞噬活性被部分挽救。在这些研究中，最佳细胞密度至关重要。当巨噬细胞以过低的密度接种时，未检测到荧光强度的变化。当巨噬细胞以高密度接种时，所有组的荧光强度迅速升高，差异不明显。在此过程中，最重要的事情之一是确保细胞密度得到优化并在实验之间保持一致。

Research

JoVE Journal

Immunology and Infection

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

科研

免疫与感染

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

猪或牛光感受器外段的细胞吞噬分析对视网膜色素上皮细胞大规模纯化

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

饱和脂肪酸诱导神经酰胺相关巨噬细胞死亡

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

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...