Name: isolation of dendritic cells from the human female reproductive tract for phenotypical and functional studies
Uploaded: 2018-03-13T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies at JoVE.com

由 NIH 资助 AI102838 和 AI117739 (CRW) 支持的研究。我们感谢理查德. Rossoll 的技术援助。流细胞分析是在 DartLab (P30CA023108-37) 和 (P30GM103415-15) Dartmouthsupported 的共享资源中进行的。

根据《赫尔辛基宣言》中所表达的原则进行了涉及人类主题的研究。研究获得了达特茅斯学院机构审查委员会和保护人类问题委员会 (CPHS) 的批准。在达特茅斯-希区柯克医疗中心 (黎巴嫩、新罕布什尔州) 接受子宫切除术的艾滋病毒阴性妇女进行手术前获得书面知情同意。经过训练的病理学家从 EM、CX 和 ECX 中选择组织样本, 没有病理性病变, 远离病理部位。血液样本是由在达特茅斯区的医学中心招募的志愿者健康捐赠者获得的。献血者是匿名的。新隔离的 EM, CX 和 ECX 组织从手术室转移到病理分离和分类之前, 转移到我们的实验室在单独的, 无菌聚丙烯管。
1. 酶消化组织
注: 使用无菌材料, 并在生物安全柜中工作。
<ol><li>用 1x HBSS 与苯酚红、100毫升青霉素-链霉素、0.35 克/升 NaHCO3和20毫米 HEPES, 制备改性的汉克平衡盐溶液 (HBSS)。</li>
	<li>制备酶鸡尾酒 (改性 HBSS, 450 U/毫升胶原酶 IV, 0.01% [重量/卷] 脱氧核糖核酸酶 I, 2 毫克/毫升 d-葡萄糖)。通过无菌0.22 µm 过滤器的解决方案。理想情况下, 在使用的当天准备消化酶, 但它可以冷冻在-20 摄氏度储存。避免反复结冰和解冻循环。</li>
	<li>用修改后的 HBSS 冲洗组织, 并将其放置在 100 x 15 毫米2培养皿中。根据可用的组织数量和使用的酶鸡尾酒量调整培养皿的尺寸 (见下面的步骤 1.4.2)。 注意: 组织的大小取决于从病理学得到的手术样本, 从1-10 克不等。</li>
	<li>用一次性手术刀和镊子进行物理处理:<ol>
<li>在组织中加入大约3毫升的消化酶鸡尾酒, 防止切割时干燥。</li>
		<li>使用镊子用手术刀稳定组织和切碎, 以增加接触消化酶鸡尾酒的组织的表面积。将组织切成小块 (< 2 毫米的一侧)。加入足够的消化酶鸡尾酒, 覆盖所有组织片 (5 毫升/克的组织)。把盖子放在培养皿上。</li>
		<li>孵育在37°c 与 5% CO2在一个旋转大约 80 rpm 为45分钟. 确保旋转速度彻底混合消化酶鸡尾酒, 不溢出或产生气泡。</li>
		<li>用4X 和10X 目标的倒置光镜可视化组织准备。 注意: 消化后, 小的组织片段和混合细胞悬浮包括可见的上皮腺体应该存在 (请参见图 1A)。</li>
	</ol>
</li>
</ol>2. 单个细胞的物理分离
<ol><li>在无菌环境中, 将拉紧的250µm 网格与持有者放入 150 x 15 毫米2培养皿中。确保网格在培养皿表面上方约0.5 厘米。</li>
	<li>用2毫升的改性 HBSS 湿网, 将所消化的组织转移到网格上。</li>
	<li>使用10毫升注射器柱塞的平坦表面, 轻轻而牢固地研磨经过筛网的经消化的碎组织。 注意: 过度磨削会破坏网格并增加细胞死亡率。这一步骤将从结缔组织和粘液中分离出上皮细胞和基质细胞 (包括免疫细胞)。</li>
	<li>一旦组织碎片被彻底分散, 提升网格和2-3 厘米以上的培养皿和冲洗与3毫升的修改 HBSS, 以恢复额外的细胞。</li>
	<li>收集细胞悬浮液。将20µm 网格与持有人在培养皿和湿与2毫升的修改 HBSS。将细胞悬浮液通过网格在培养皿上方2-3 厘米的网眼上倒入, 并用6毫升的改性 HBSS 冲洗。 注意: 这一步将分离的上皮片, 这是保留在顶部, 从单一的细胞, 通过网格。流动是一种混合细胞悬浮液, 包括免疫细胞和基质成纤维细胞。</li>
	<li>收集混合细胞悬浮液和离心机在 500 x g 10 分钟内吸液体和并用重悬颗粒在4毫升的改性 HBSS 的密度梯度离心。</li>
	<li>将混合细胞悬浮在3毫升以上的聚蔗糖溶液中, 在室温下离心机以 500 x g 为30分钟, 无刹车。收集聚蔗糖溶液顶部的白带, 并用 PBS 清洗。 注: 本部分为富含免疫细胞的富含混合细胞悬浮液, 含有部分基质成纤维细胞。</li>
</ol>3. 去除死细胞
注: 如果密度梯度离心后富集混合细胞悬浮液中有很大比例的死细胞 (> 20%) 存在, 建议用磁性珠子去除死电池。死细胞需要被去除, 因为它们可能干扰正磁珠选择过程。
<ol><li>使用10X 目标的光镜中的 hemocytometer 计算细胞数。在富集混合细胞悬浮液中旋转所有细胞 (500 x g, 10 分钟, 4 °c)。完全吸入并丢弃上清液。根据制造商的说明, 添加100µL 的死细胞去除珠, 每 1 x 107总细胞 (活和死)。室温孵育15分钟。</li>
	<li>将30µm 过滤器放在磁柱上, 应用细胞悬浮, 并允许珠子标记的细胞通过磁柱坠落。</li>
	<li>如建议 (3 毫升), 用死细胞去除缓冲器冲洗柱。收集包含活细胞的流。在图 1B中显示了死移除后单元格恢复的范围。 注: 这些细胞可用于表型鉴定研究的流式细胞仪染色 (5 节) 或继续 DC 隔离 (4 节)。</li>
</ol>4. 用正磁珠选择直流净化
<ol><li>在死细胞清除 (步骤 3.2), 旋转细胞向下, 移除上清, 并并用重悬细胞颗粒在80µL 磁选择缓冲区每 1 x10 7 细胞 (PBS, 0.5% 热灭活人 AB 血清 (HS), 2 毫米 EDTA)。</li>
	<li>为积极选择 DC 的人口, 添加 CD1a 或 CD14 磁性珠根据制造商的指示 (20 µL 磁性珠每 1 x 107单元格)。孵育15分钟在 4°c.
</li>
	<li>用磁选缓冲器、向下旋转和完全删除缓冲区来清洗单元格。并用重悬最小的500µL 磁选缓冲器的细胞。</li>
	<li>将该列附加到磁铁上, 并在该列的顶部添加一个30µm 滤镜, 以保留剩余的单元格块。按照推荐的磁选缓冲器冲洗过滤器和柱。</li>
	<li>将单元格悬置应用于该列。用磁选缓冲器冲洗3次。</li>
	<li>将该列转移到15毫升管, 添加5毫升的磁选缓冲器, 并应用柱塞从列中释放选定的单元格。</li>
	<li>要提高纯度, 请使用新列对恢复的单元格重复步骤 4.4-4.6。在图 1C中显示磁珠选择后的单元恢复范围。</li>
</ol>5. 细胞纯度的评估: 流式细胞仪和显微术
<ol><li>流式细胞术的抗体染色:<ol>
<li>并用重悬100µL 中的 1 x10 6 单元格, 包含20% 热灭活 HS, 以阻止 Fc 受体, 并在冰上孵化10分钟。</li>
		<li>添加所需的荧光标记抗体和用于鉴定死细胞的试剂 (例如, 见第6.5 节)。设置荧光减一 (FMO) 控制建立闸门, 并使用补偿珠准备单一的污点控制, 以补偿。使用的抗体示例在表 1中给出。孵育20分钟, 4 摄氏度, 免受光照。</li>
		<li>用2毫升的磁选缓冲器清洗细胞。 注: EDTA 的存在对于避免细胞结块形成流细胞分析是很重要的。向下旋转并丢弃上清。</li>
		<li>通过增加每管100µL 2% 多聚甲醛溶液来修复细胞。</li>
		<li>通过流式细胞仪10、11来分析示例。</li>
	</ol>
</li>
	<li>旋转该列 (例如、cytospin) 并使用姬姆萨染色来分析形态学:<ol>
<li>并用重悬200µL 磁选缓冲器中的细胞。</li>
		<li>在旋转柱中加载, 旋转5分钟。让幻灯片完全干燥。</li>
		<li>用100% 甲醇浸泡2分钟, 用去离子水冲洗滑梯, 使其风干。</li>
		<li>执行标准 Wrights 姬姆萨染色。用红血球覆盖细胞, 孵育10分钟, 用去离子水冲洗, 使其干燥。用 Wrights Geimsa 染色盖住细胞, 孵育1分钟, 用去离子水冲洗, 风干。</li>
		<li>用倒置显微镜 (图 2A, 40X 目标) 检查准备工作。</li>
	</ol>
</li>
</ol>6. 同种异体刺激法评价 DC 细胞功能
<ol><li>解冻冷冻外周血单个核细胞 (PBMCs)<ol>
<li>温暖细胞培养媒介与 10% HS 到37°c。</li>
		<li>将 cryotube 放在37摄氏度的水浴中解冻 PBMCs, 直到少量冰冻细胞介质留在 cryotube。在无菌环境中, 将 cryotube 的内容转移到10毫升的预热细胞培养培养基中, 10% HS 在15毫升管中。</li>
		<li>离心机在〜 500 x g 7 分钟去除介质, 分散颗粒, 并用重悬 PBMCs 10 毫升细胞培养基与 10% HS, 并离心细胞。</li>
		<li>第三次重复洗涤步骤。计数单元格。</li>
	</ol>
</li>
	<li>天真的 T 细胞选择:<ol>
<li>使用阴性选择磁性抗体细胞选择套件根据制造商的协议隔离幼稚的 T 细胞。</li>
		<li>隔离后, 检查纯度使用 CD45RA 和 CCR7 抗体。 注意: 典型的纯度是 > 99% 天真的 T 细胞。</li>
	</ol>
</li>
	<li>天真的 T 细胞增殖染料染色:<ol>
<li>制备600µL 细胞增殖染料的2x 溶液, 保护染料不受光照射。</li>
		<li>用 PBS 清洗天真的 T 细胞2x。并用重悬细胞在500µL PBS。</li>
		<li>在安全柜中轻轻涡流细胞时, 将500µL 的增殖染料添加到天真的 T 细胞中。</li>
		<li>在37°c 中孵化细胞10分钟.
</li>
		<li>通过添加5毫升细胞培养培养基与 10% HS 细胞来停止细胞标记。在冰上孵化5分钟, 免受光照。</li>
		<li>离心细胞在 ~ 500 x g 7 分钟, 吸入介质, 并并用重悬细胞培养基4°c 与 10% HS。重复两次, 总共3洗。在最后一次离心之前, 取一个整除的细胞计数。 注意: 通常, 最终天真的 T 细胞数将介于初始 PBMC 细胞计数的5% 和15% 之间。</li>
		<li>并用重悬细胞培养培养基与 10% HS 在所需的细胞浓度。</li>
	</ol>
</li>
	<li>异基因天真的 T 细胞共培养:<ol>
<li>将孤立的 dcs 和天真的 T 细胞板在一个96井, 圆底板, 在1:15 的比例的 DCs 到 T 细胞。确保 DCs 的最佳数字范围介于3000和5000单元格之间。</li>
		<li>离心板在〜 500 x g 3 分钟, 以确保细胞位于井的中心。细胞对细胞的接触对于正确的信号发生是很重要的。</li>
		<li>孵育37°c 6 天。通过显微术 (图 3A) 监视在油井中出现的细胞簇。</li>
	</ol>
</li>
	<li>流式细胞仪染色评估增殖:<ol>
<li>6天后, 细胞可以通过流式细胞术进行检查。对流式细胞术的共培养进行染色。</li>
		<li>准备一个2x 溶液的黄色活性染料 (最大排放 572 nm) 在 PBS。 注意: 使用无血清 PBS 是重要的, 因为血清蛋白干扰染料)。</li>
		<li>用 500 x g 的细胞离心出5分钟的板。</li>
		<li>去除100µL 的上清。 注: 在这一点上收集和储存上清, 以分离分析可溶性因素。</li>
		<li>用 pbs 两次冲洗细胞: 在 pbs 中加150µL/井, 离心机在 ~ 500 x g 处5分钟, 用吸管去除150µL 的上清液。确保每个井中都有50µL 的上清液。</li>
		<li>在每个井中添加50µL 的2x 活性染料。室温孵育10分钟, 免受光照。</li>
		<li>在孵化过程中, 准备好所需抗体标记的总混合物。例如: CD3 APC/Cy7、CD4 PE、CD8 FITC、等.
</li>
		<li>将抗体混合物添加到每个井中。室温孵育10分钟, 免受光照。</li>
		<li>2x. 用 pbs + 2% hs 冲洗单元格: 增加150µL/井的 pbs + 2% hs。离心机在〜 500 x g 5 分钟, 用吸管去除150µL 的上清液。</li>
		<li>通过添加100µL/井2% 的准甲醛来修复细胞。如果需要: 将细胞和介质转移到聚丙烯流式细胞术管。管子可以保持在4°c 和保护从光, 直到流动细胞分析。</li>
		<li>在流式细胞仪中读管。</li>
	</ol>
</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+and+susceptibility+to+HIV+infection+of+CD4++Th17+cells+in+the+human+female+reproductive+tract.">Phenotype and susceptibility to HIV infection of CD4+ Th17 cells in the human female reproductive tract.</a> Mucosal Immunol. 7 (6), 1375-1385 (2014).</li><li>Shen, Z., Rodriguez-Garcia, M., Ochsenbauer, C., Wira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+immune+cells+and+infection+by+HIV+in+human+ovarian+tissues.">Characterization of immune cells and infection by HIV in human ovarian tissues.</a> Am J Reprod Immunol. , (2017).</li></ol>

作者没有什么可透露的

粘膜 DCs 是一种罕见的细胞群, 受组织环境的强烈影响, 一旦进入组织3, 就会改变其表型和功能。虽然血源性 DCs 是一个非常有用的模型, 但它们并不完全代表在组织中发现的 DC 种群的多样性。因此, 要了解粘膜 DCs 的独特特点, 必须从组织中分离出原细胞。在首次登记中, 从不同解剖部位分离出 DCs, 为研究直流功能在体外提供了机会, 并比较了 dc 子集和解剖位置。
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首次登记税具有保护病原体的双重功能, 同时允许植入和妊娠1。为了做到这一点, 首次登记税是分开的, 每个解剖区域显示独特的组织学, 免疫学和功能特征1。
DCs 存在于粘膜表面, 与肺、肠道和生殖道中的微生物接触, 并为潜在病原体提供免疫监测2。DCs 有独特的能力, 主要天真的 T 细胞和触发自适应免疫应答3。在首次登记税中, DCs 也专门用于容忍外国抗原, 如精子和发育中胎儿的抗体, 以允许成功怀孕4。因此, 根据位置, DC 表型和功能将是不同的。众所周知, DCs 受到组织环境的强烈影响, 因此它们的数量、表型和功能都被其驻留3的组织环境所修改。因此, 为瞭解首次登记税区议会在首次登记税中的传染病、怀孕及癌症所扮演的角色, 需要研究居民 dcs, 因为血液衍生的 dc 模型不足以解决首次登记税组织所发现的监管复杂性。
由于黏膜组织中的细胞数量少, 人体组织样本难以获得, 人体组织 DCs 的特性具有挑战性。使用免疫组化5、6在首次登记税中研究了 DCs, 它通知了组织内的细胞位置, 但排除了功能性研究, 并且限制了可以分析的识别细胞标记的数量。一次。此外, 已开发了用于流细胞分析的单个单元隔离协议7、8、9。其中一些协议利用 DCs 的迁移能力来隔离那些迁移的单元格。这些方法通常需要夜间孵化和选择的活化 dcs, 但不允许对 dcs 的研究在稳定的状态。
在这里, 使用子宫切除标本, 我们优化了一个协议, 以隔离 DCs 从不同解剖地点的首次登记税, 子宫内膜 (EM), 宫颈管 (CX) 和 ectocervix (ECX), 这使得表型鉴定和功能分析。使用非蛋白水解酶消化协议, 我们可以立即进行组织消化后细胞分离和流动细胞表征, 而不激活细胞。采用多色流式细胞术和对低细胞数进行功能研究, 可以识别和表征首次登记的不同部位的 DCs 稀有子集。

<table><tbody><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Hyclone</td><td>SH30015.03</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>HEPES (1M)</td><td>Hyclone</td><td>15630-080</td><td></td></tr><tr><td>Collagenase IV</td><td>Sigma</td><td>C5138</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Worthington Biochemical</td><td>LS002140</td><td></td></tr><tr><td>D-glucose</td><td>Sigma Aldritch</td><td>50-99-7</td><td></td></tr><tr><td>0.22 um Stericup 500 mL&amp;nbsp; filter</td><td>Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>100mm x 15mm polystyrene petri dish</td><td>Fisherbrand</td><td>FB0875712</td><td></td></tr><tr><td>150mm x 15mm polystyrene petri dish</td><td>Fisherbrand</td><td>FB0875714</td><td></td></tr><tr><td>150mm x 25mm polystyrene dish</td><td>Corning</td><td>430599</td><td>Treated cell culture dish</td></tr><tr><td>Isotemp Incubator</td><td>Fisher Scientific</td><td>FICO3500TABB</td><td>5.0% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>American Rotator V</td><td>American DADE</td><td>R4140</td><td></td></tr><tr><td>250 um nylon mesh</td><td>Sefar</td><td>03-250/50</td><td></td></tr><tr><td>20 um nylon mesh</td><td>Sefar</td><td>03-20/14</td><td></td></tr><tr><td>Beckman GS-6R Centrifuge</td><td>Beckman</td><td>358702</td><td></td></tr><tr><td>X-VIVO 15 with Gentamicin L-Gln, Phenol Red, 1 L</td><td>Lonza</td><td>04-418Q</td><td></td></tr><tr><td>Human AB Serum</td><td>Valley Biomedical</td><td>HP1022</td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma Aldritch</td><td>10771</td><td></td></tr><tr><td>Phosphate Buffer Solution (PBS)</td><td>National Diagnostics</td><td>CL-253</td><td>pH 7.4</td></tr><tr><td>Dead Cell Removal Kit</td><td>Miltenyi Biotec</td><td>130-090-101</td><td></td></tr><tr><td>Pre-separation filter (30um)</td><td>Miltenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>LS column</td><td>Miltenyi Biotec</td><td>130-042-410</td><td></td></tr><tr><td>Quadro MACS Separator</td><td>Miltenyi Biotec</td><td>130-090-976</td><td></td></tr><tr><td>MACS multi-stand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>EDTA</td><td>USB</td><td>15694</td><td></td></tr><tr><td>CD1a Microbeads, human</td><td>Miltenyi Biotec</td><td>130-051-001</td><td></td></tr><tr><td>CD14 Microbeads, human</td><td>Miltenyi Biotec</td><td>130-050-201</td><td></td></tr><tr><td>eFluor 670 cell proliferation dye</td><td>eBiosciences</td><td>65-0840-85</td><td></td></tr><tr><td>96 well round bottom plate</td><td>Falcon</td><td>9/8/2866</td><td></td></tr><tr><td>Zombie yellow viability dye</td><td>Biolegend</td><td>423104</td><td></td></tr><tr><td>CD3 APC/Cy7, anti-human</td><td>Tonbo Biosciences</td><td>25-0038-T100</td><td></td></tr><tr><td>CD4 PE, anti-human</td><td>eBiosciences</td><td>12-0048-42</td><td></td></tr><tr><td>CD8a FITC, anti-human</td><td>Tonbo Biosciences</td><td>35-0086-T100</td><td></td></tr><tr><td>Gallios Flow Cytometer</td><td>Beckman Coulter Life Sciences</td><td>B43618</td><td>10 color, VBR</td></tr><tr><td>MACSquant Analyzer 10</td><td>Miltenyi Biotec</td><td>130-096-343</td><td>8 color, VBR</td></tr></tbody></table>

isolation of dendritic cells from the human female reproductive tract for phenotypical and functional studies

人树突状细胞 (DCs) 在黏膜组织中的特征是挑战, 由于难以获得样本, 和低数量的 DCs 目前每组织。然而, 由于在组织环境中对 dcs 的表型和功能进行了修改, 因此有必要对组织驻留 DC 种群进行分析, 因为血源 dcs 不完全反映了组织中 dcs 的复杂性。在这里, 我们提出一个协议, 以隔离 DCs 从人类女性生殖道 (首次登记) 使用子宫切除标本, 允许表型鉴定和功能分析。该协议包括组织消化生成单个细胞混合细胞悬浮, 其次是正磁珠选择。我们的组织消化协议不切割表面标记, 这允许表型鉴定和功能分析的 DCs 在稳定的状态, 没有夜间孵化或细胞活化。该协议可用于隔离其他免疫细胞类型或隔离 DCs 与其他组织。

组织消化后, 可以观察上皮片和腺体的释放情况, 如图 1A所示;这是一个积极的控制, 表明酶消化是成功的。在图 1B和图 1C中, 每克组织恢复的可生存细胞总数和 DCs 数量也分别显示。在密度离心12,13之前, 免疫细胞代表5-30% 的基质细胞。图 2显...

Watch this Scientific Journal Video about Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies at JoVE.com

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

本文介绍了一种从人类女性生殖道的不同解剖间隔分离纯化树突状细胞的方法, 以评价其表型鉴定和功能特征。该方法可用于隔离其他粘膜组织中的其他免疫细胞或树突状细胞。

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

isolation-dendritic-cells-from-human-female-reproductive-tract-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.4K 次观看.  Geisel School of Medicine at Dartmouth. 本文介绍了一种从人类女性生殖道的不同解剖间隔分离纯化树突状细胞的方法, 以评价其表型鉴定和功能特征。该方法可用于隔离其他粘膜组织中的其他免疫细胞或树突状细胞。

视频: Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

科研

癌症研究

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

免疫与感染

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

生物学

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.