Name: experimental analysis of apoptotic thymocyte engulfment by macrophages
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邵实验室的研究得到了医学院研究创新奖和辛辛那提大学内科初级教师试点奖的支持, 并获得了 NIDDK/nih 颁发的 DK K01_095067。

实验小鼠在我们的小鼠群体中进行了培育和维持。所有动物工作都是根据辛辛那提大学动物护理和使用机构委员会 (IACUC) 的指导方针进行的。
1. CFSE 标记凋亡胸腺细胞的制备
<ol><li>用 CO2 吸入使两只天真的 C57/B6 小鼠安乐死 10分钟, 并解剖打开胸腔, 用弯曲的细尖钳将胸腺取出 (拔出) 到含有 RPM1640 培养基的10毫升的组织培养培养皿中。</li>
	<li>通过将整个胸腺磨成显微?...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+cancer:+from+pathogenesis+to+treatment.">Apoptosis in cancer: from pathogenesis to treatment.</a> Journal of Experimental and Clinical Cancer Research. 30, 87 (2011).</li><li>Baig, 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=Potential+of+apoptotic+pathway-targeted+cancer+therapeutic+research:+Where+do+we+stand.">Potential of apoptotic pathway-targeted cancer therapeutic research: Where do we stand.</a> Cell Death and Disease. 7, 2058 (2016).</li><li>Poon, I. K., Lucas, C. D., Rossi, A. G., Ravichandran, K. 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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=The+anticoagulation+factor+protein+S+and+its+relative,+Gas6,+are+ligands+for+the+Tyro+3/Axl+family+of+receptor+tyrosine+kinases.">The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases.</a> Cell. 80 (4), 661-670 (1995).</li><li>Linger, R. M., Keating, A. K., Earp, H. S., Graham, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAM+receptor+tyrosine+kinases:+biologic+functions,+signaling,+and+potential+therapeutic+targeting+in+human+cancer.">TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer.</a> Advances in Cancer Research. 100, 35-83 (2008).</li><li>van der Meer, J. 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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+for+inducing+apoptosis.">Methods for inducing apoptosis.</a> Methods in Molecular Medicine. 102, 115-128 (2004).</li><li>Progatzky, F., Dallman, M. 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细胞凋亡是一个高度保守的细胞死亡过程, 涉及许多信号级联, 并诱导蛋白质的表达, 分泌和运输。细胞凋亡常与细胞形态变化有关 17。凋亡细胞主动释放细胞因子和趋化因子, 吸引吞噬细胞迁移到该位点, 并在严格控制下启动吞没过程18。另一方面, 坏死细胞死亡释放引发炎症反应的危险信号1。凋亡细胞的有效或长时间清除会导致这些细胞的继?...

我们的身体每天产生1-10 个凋亡细胞。如此大量的凋亡细胞必须以免疫反应保持静止的方式清除。为了确保及时清除凋亡细胞, 多种组织驻体细胞和循环细胞发展出吞没凋亡细胞的机制 1。细胞凋亡的功能失调调节与各种炎症性疾病的发生和进展有关, 自身免疫 2。细胞凋亡在癌症发展的发病机制及其对常规治疗的抵抗力3,4中也起着至关重要的作用。去除凋亡细胞通常会促进抗炎反应, 这可能与免疫耐受5有关。凋亡细胞清除的干扰推动了自我免疫, 并有助于人类和小鼠全身自身免疫性疾病的发展。
当细胞发生凋亡时, 它们将磷脂酰丝氨酸 (PtdSer) 从内小叶暴露到膜的外小叶。然后, PtdSer 将通过表面受体被吞噬细胞识别。已经发现了十几个受体, 以识别和/或促进凋亡细胞的吞没。一般情况下, 至少有三种类型的表面受体参与凋亡细胞清除: 网络受体, 识别凋亡细胞;发痒的受体, 启动吞没;陪护受体, 促进整个过程7。TAM 受体酪氨酸激酶 (TAM Rtk) 由tyro-3、 axl 和mer 组成, 主要由免疫系统的骨髓细胞表达8。TAM Rtk 的主要功能是作为连接受体, 促进凋亡细胞和碎片的吞噬性去除。本组多年来一直在自身免疫环境下研究 TAM 介导的凋亡细胞清除。维生素 k 依赖性蛋白生长抑制特定蛋白 6 (gas6) 和蛋白 s (pros) 结合并激活 tam 受体9,10。Gas6 产生于心脏、肾脏和肺部。P s 主要是在肝脏中产生的11。TAM 识别凋亡细胞的方式是, Gas6/ProS 的 n 端与凋亡细胞上的 PtdSer 结合, Gas6/ProS 的 c 端与锚定在吞噬细胞表面的 TAM 受体结合。与其他受体一起, 凋亡细胞吞没发生 12。虽然尔可以与配体 ProS 和 Gas6 结合, 但我们发现 Gas6 似乎是 em 介导的凋亡细胞巨噬细胞吞噬的唯一配体, 而这可以被抗莫抗体13阻断.巨噬细胞是专业的吞噬细胞。巨噬细胞快速清除凋亡细胞对于抑制细胞内抗原的炎症和自身免疫反应具有重要意义。莫氏受体酪氨酸激酶是巨噬细胞吞没和凋亡细胞的有效清除14的关键。在小鼠脾脏中, Maer 主要表现为边缘区和有形体巨噬细胞13。
本文介绍的协议描述了诱导细胞凋亡的基本方法, 并演示了检测细胞凋亡过程和效率的方法。这些方案可以很容易地适应研究其他细胞类型的自泡增多症, 在不同来源的凋亡细胞吞没。

<table border="0" cellpadding="0" cellspacing="0" style="width:832px;" width="832">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Ack lysing buffer</td>
			<td style="width:131px;">GIBCO</td>
			<td style="width:135px;">A10492</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Annexin V/7-AAD</td>
			<td style="width:131px;">BD Pharmingen</td>
			<td style="width:135px;">559763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Anti-Mer antibody</td>
			<td style="width:131px;">R&#38;D Systems</td>
			<td style="width:135px;">BAF591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">CD11b-PE (clone M1/70)</td>
			<td style="width:131px;">BD Pharmingen</td>
			<td style="width:135px;">553311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">CFSE</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:135px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">DMSO</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:135px;">D-2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">EDTA (0.5 mM)</td>
			<td style="width:131px;">GIBCO</td>
			<td style="width:135px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">FACS tubes</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">352017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Frosted slides</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">12-552-343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Horse Serum (Heat-inactivated)</td>
			<td style="width:131px;">Invitrogen</td>
			<td style="width:135px;">26050088</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Lidocaine</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:135px;">L-5647</td>
			<td style="width:275px;">Prepare 1% buffer in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">PBS, 1x</td>
			<td style="width:131px;">Corning</td>
			<td style="width:135px;">21040CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">RPMI-1640</td>
			<td style="width:131px;">Corning</td>
			<td style="width:135px;">10040CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">RXDX-106</td>
			<td style="width:131px;">Selleck Chemicals</td>
			<td style="width:135px;">CEP-40783</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:292px;">Staurosprine (100mg)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">BP2541-100</td>
			<td style="width:275px;">Add 214.3 ml of DMSO into 100mg to make 1mM stocking solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:292px;">Thioglycolate Medium Brewer Modified</td>
			<td style="width:131px;">BD Biosciences</td>
			<td style="width:135px;">243010</td>
			<td style="width:275px;">Prepare 3% thioglycolate buffer in 1&#180;PBS, autoclaved, and store in the dark for 3 months.</td>
		</tr>
	</tbody>
</table>

experimental analysis of apoptotic thymocyte engulfment by macrophages

细胞凋亡是一个自然的过程, 在胚胎发育、稳态调节、免疫耐受诱导和炎症的治疗中起着至关重要的作用。体内凋亡碎片的积累可能会引发慢性炎症反应, 随着时间的推移导致全身自身免疫性疾病。凋亡细胞清除障碍与各种自身免疫性疾病有关。凋亡清除是一个复杂的过程, 在生理条件下很少检测到。它涉及丰富的表面感受器官和信号分子。研究凋亡细胞清除过程提供了深刻的分子机制和随后的生物反应, 这可能导致新的治疗方法的发展。在这里, 我们描述了诱导凋亡胸腺细胞, 腹腔巨噬细胞的制备, 以及流式细胞仪和显微镜分析凋亡细胞清除的协议。所有细胞在一定阶段都会发生凋亡, 许多体内和循环细胞可以吸收凋亡碎片。因此, 这里描述的协议可用于许多应用, 以表征凋亡细胞结合和摄入的许多其他细胞类型。

腹膜巨噬细胞介导的凋亡胸腺细胞吞没分析.按照该方案的描述, 制备并联合培养腹腔巨噬细胞和凋亡细胞。巨噬细胞在冰上被 pe 共轭抗 CD11b 抗体分离并染色20分钟。然后用流式细胞仪清洗和处理巨噬细胞。如图所示, 当培养物中没有添加凋亡细胞时, 右下角象限中没有 CFSE 阳性巨噬细胞 (图 1 a 和图 2, 第一面板)。硫勒糖刺激的?...

Watch this Scientific Journal Video about Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages at JoVE.com

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

在这里, 我们提出了一个方案, 以编制凋亡胸腺细胞和腹膜巨噬细胞, 并分析了细胞内吞的效率和特定的抑制介导的抑制凋亡胸腺细胞吞没。该协议在包括人工珠子和细菌在内的其他颗粒的细胞介导清除中有着广泛的应用。

巨噬细胞吞噬胸腺细胞吞噬的实验分析

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

我们的身体每天产生100亿到100亿个凋亡细胞。这些电池的高效清除有助于清除细胞碎片并维持库存。这种方法可用于许多应用，以描述凋亡细胞结合和摄入许多其他噬细胞类型。演示这个程序的将是我实验室的高级技师郑宇轩。要收获胸腺细胞，打开一只天真的C57黑色6鼠标的胸腔后，使用弯曲的细尖钳拉出胸腺。将器官放入含有10毫升RPMI 1640中等的组织培养盘中。将整个胸腺磨碎在两个显微镜幻灯片的磨砂端上，并通过 70 微米细胞过滤器将产生的组织悬浮液过滤到 50 毫升管中。通过离心收集胸腺细胞，并在40毫升中重新暂停颗粒。计数后，再次离心细胞，每50毫升管20毫升新鲜PBS中，将2倍10重新向8个细胞重新暂停。将细胞与5微摩尔CFSE标注每管20毫升PBS。在室细胞孵育时，在室温下将胸腺细胞孵育不超过两分钟，然后反转管子两到三次，以混合。在孵育结束时，停止与10毫升热灭活马血清的反应，通过离心收集细胞。将标记的细胞重新用 40 毫升新鲜 PBS 中重新暂停，用于计数和离心，然后用 40 毫升的介质进行额外的洗涤。中等洗涤后，将胸腺细胞以每毫升组织培养培养力的7倍10重新暂停至6个细胞，并在100毫米组织培养皿中播种细胞。然后以一微摩尔最终浓度将葡萄球菌加入细胞培养，并将板放在组织培养培养箱中四小时。对于腹膜巨噬细胞分离，在第5天打开腹部皮肤而不干扰腹腔内，将两只C57黑6小鼠注射一毫升3%的白细胞酸酯，而不干扰腹腔。要冲洗腹腔，请使用装有 18 量针的 10 毫升注射器快速将 10 毫升洗涤缓冲液推入腔中，然后缓慢吸气洗涤缓冲液以收集到 50 毫升管中。用 PBS 清洗近体熔岩两次。将足科巨噬细胞以每毫升组织培养中浓度的2倍10重新吸收至6个细胞。下一种子500微升的巨噬细胞进入24井板的每个井，并放置板在组织培养箱两个小时。在孵育结束时，吸上经剂去除漂浮细胞，并在每井中加入500微升组织培养。立即将培养物中500微升的6个胸腺细胞的0至12倍10添加到培养物的每个井中，并将板放在组织培养培养箱中4小时。在孵育结束时，用新鲜的PBS清洗每井两次，以去除任何自由浮动凋亡细胞，然后用染色缓冲液洗一次。接下来，用200微升染色缓冲液为板装巨噬细胞贴上标签，每井辅以适当的抗CD11b抗体，将板在4摄氏度下放置20分钟。在这个具有代表性的实验中，高达30%的硫化细胞刺激的内酮性巨噬细胞在CFSE通道中显示出一个阳性信号，表明这些噬菌体摄入了CFSE标记的凋亡细胞。对凋亡细胞的巨噬细胞吞没的微观观察对于研究巨噬细胞摄入凋亡细胞的能力至关重要，因为CFSE正巨噬细胞在右下象限中扩散，因为强度不同，表明单个巨噬细胞内的凋亡细胞数量不同。凋亡细胞与巨噬细胞的比例越高，不仅增加了摄入凋亡细胞的巨噬细胞数量，而且提高了巨噬细胞摄入更多凋亡细胞的能力。在另一组实验中，当CFSE将标记的凋亡胸腺细胞添加到巨噬细胞培养物中4小时，表明这些巨噬细胞是噬菌体时，大约15%的巨噬细胞成为CFSE阳性。Mer抗体以剂量依赖的方式阻断巨噬细胞增多症，在目前环境中，整体阻塞可能占到紫外细胞病效率的30%左右。此外，新批准的TAM受体抑制剂以剂量依赖的方式衰减巨噬细胞增多，浓度高达约100纳米摩尔。始终记得检查凋亡细胞的百分比，因为这个数字直接影响到肺功能化的效率。噬菌体巨噬细胞可以通过西方的污点进一步分析，以研究由吞没过程和显微镜调节的信号分子，以研究吞没的动力学。

Research

JoVE Journal

Immunology and Infection

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

科研

免疫与感染

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...