这项工作是由加拿大安大略省癌症研究所（OICR），Terry Fox基金会和美国神经外科医师协会。

1。对脑肿瘤的组织文化<ol><li> 200μL解冻Liberase的（罗氏应用科学部）15毫升的人工脑脊液（ACSF 见表1），并放入37℃水浴。 liberase TM是用于解离主要的组织样本，以及培养tumorspheres的混合的蛋白水解酶。胰蛋白酶-EDTA不同中，Liberase方法保留了表面抗原CD133。对于一个约0.5厘米3的组织样本，我们用200微升Liberase。如果组织是较小的，我们用100微升。 </li><li>带上的氯化铵溶液（干细胞技术）到室温。氯化铵溶液中轻轻地溶解红血细胞与其他细胞的影响最小。它不包含一个固定液。 </li><li>在无菌生物安全柜中，加入5毫升的脑脊液标本的容器，涡流冲洗组织，然后移液管关闭。此步骤有助于去除血红细胞（RBC）。 </li><li>脑肿瘤组织转移到无菌的100毫米的Petri二叔SH。 </li><li>使用剪刀或手术刀和镊子，分解组织浆的一致性。 </li><li>收集样品进入管预热的脑脊液Liberase的定期用10毫升的移液管或镊子和转让片段。 </li><li>放置在培养箱摇床（每分钟30转），并设定为37℃，持续15分钟。 </li><li>过滤器的组织裂解液通过70μm的细胞过滤网入50毫升的Falcon管中。 </li><li>附带滤液，在280×g离心5分钟。 </li><li>仔细移除上清液和评价所得到的细胞沉淀的大小和颜色:粒料是粉红色或红色，表明增加的红血细胞的数目。 </li><li>在1ml的PBS重悬沉淀。 </li><li>添加适当量的氯化铵溶液（4-12毫升），根据颗粒大小和红细胞污染（氯化铵溶液是非常温和的增加量不是有害细胞红细胞以外）。 </li><li>在室温下孵育5分钟。 </li><li>自旋细胞下降，在280×g离心5分钟。 </li><li>用10ml无菌PBS洗涤一次。 </li><li>重悬在5毫升NSC完全培养基（ 表2），并转移至超低结合60毫米组织培养板（Corning）。我们采用超低约束力的培养板共价键合的水凝胶表面的亲水性和电中性的，以减少细胞的粘附介导的分化。 </li></ol>文化的最初几天中， 不改变介质上，只用1-2毫升媒体，然后继续观察文化和变革媒体的媒体时的颜色变成微黄色。 2。 Tumorsphere解离流式细胞仪分析<ol><li>评估tumorspheres显微镜下:如果球体大小是&gt; 100微米，解离被推荐为在中心，较大的球可能成为坏死。 </li><li>转移至15毫升锥形管中的文化。 </li><li>加入2-3毫升无菌PBS彻底冲洗板，锥形管。 </li><li> 280×g离心5分钟离心。 </li><li>去除上清，重悬在1 - 2毫升无菌PBS。 </li><li>加入10微升Liberase。 </li><li>在37℃水浴孵育3分钟。删除和视觉评估暂停，如果看到多个团块，轻轻捣碎使用1000微升移液器吸头。 </li><li>如果丛生仍然存在，潜伏期为1-2分钟。 </li><li>洗涤细胞，加入5毫升无菌PBS中，并在280×g离心5分钟离心。 </li><li>去除上清，重悬在500-1000μl无菌PBS +2 MM EDTA。 </li><li>评估使用台盼蓝染色的细胞数和存活率。 </li><li>调整细胞计数为100万/ ml的PBS +2 MM EDTA。 </li></ol> 3。表面染色<ol><li> 100μl的1×10 6 / ml的细胞悬浮液，每次测试传输12x75毫米的流量管的。 </li><li>加入适量的抗体。匹配的抗体同种型控制应该被用于各种抗体（ 见表3）。 </li><li>在冰上孵育30分钟。 </li><li>每个流管中加入2ml PBS +2毫米EDTA。 </li><li>在200 XG离心4分钟，滗和印迹。 </li><li>在300μlPBS +2 MM EDTA重新悬浮颗粒。 </li><li>每个管中加入10μl7AAD生存能力染料和至少15分钟，在冰上孵育。 </li><li>用流式细胞仪分析。 </li></ol> 4。流式细胞仪采集和分析的具体内容的流数据的采集和分析仪器依赖。代表阴性样品的运行和仪器设置为正向（大小）和侧面（粒度）分散。这种分散的模式，允许最终用户查看所有细胞样品中，包括碎片。通常是一个区域周围绘制所关注的细胞。 （ 图4a）的设置，然后建立了荧光检测器，定位阴性细胞内的第一个十年AFluorescence强度的情节。当使用一个以上的染料或荧光染料，单染色控件必须运行建立颜色补偿值（减法干扰荧光发射）。运行时的活细胞，如7-AAD（7 -氨基放线菌素D - 655激发488/emission）被使用并排除死细胞（ 图4b）的第二区域绘制生存能力染料。这些区域都被施加到样品的任何进一步的分析。 5。自我更新的含量关键的是极大地促进通过克隆球体生长的测定是在体外试验的自我更新的能力，极限稀释法。 Tumorspheres分离，在的稀释降低到一个单细胞，每孔分布，计算和随后的球体形成率超过7天中文化。第7天，井的百分比不含有球体为每个细胞电镀密度（F 0）的计算和绘制对每孔的细胞的数量（x）的。形成在每一个孔的至少一个tumorsphere所需的细胞的数目来确定的点，在该点的线穿过0.37的水平（F 0 = e-x的 ）。在一个泊松分布的细胞，F 0 = 0.37对应于一个细胞每孔的稀释，因此，该计算（37％相交）反映人口（ 图5）在整个细胞集落形成的干细胞的频率。 6。单细胞RT-PCR 不同人群的单细胞进行排序Beckman Coulter公司MoFlo的XDP反应的AmpliGrid幻灯片的网站（Beckman Coulter公司猫＃AG480F）。单细胞RT-PCR根据制造商的说明使用AmpliGrid单一步法RT-PCR系统（Beckman Coulter公司猫＃OAX04515）。简要地说，一个单一的单元被存入每个反应部位的AmpliGrid幻灯片的存在下，在每一个单元格在显微镜下观察反应现场确认。后立即进行排序，以防止样品受到反转录（RT）。 RT预混新鲜配制，根据试剂盒说明书（ 表4）。我们使用0.03微升25μM的随机引物（Promega）中，每个反应; 1μl反应混合液添加到每个反应部位的中心。这立即涂覆与5μl的密封解决方案（Beckman Coulter公司猫＃OAX04503）。使用一个AmpliSpeed​​的滑动循环仪（Beckman Coulter公司;猫＃OAX04101），幻灯片孵育在25℃下为10分钟，42℃下为10分钟，和55℃下45分钟。反转录反应后，3微升DNA酶/无RNase的水被添加到每个反应位点，并且转移到PCR管（1管/部位）整个混合物。在PCR板，反应体积为10μl的用于定量PCR:8微升定量PCR的master mix（5微升的SYBR Green（广达），1微升的水混合，1微升10μM的正向和反向引物混合物）加2及万亩; L的RT混合物。对于实时定量，样品在BioRad公司循环仪运行使用下面的程序:95℃10分钟，45个循环的95℃30秒，60℃60秒，72℃60秒，随后通过10分钟在72℃，熔解曲线分析。使用:OPTICON监视器3（BioRad公司）测定Ct值。每个单元格可以评估三种基因，包括作为管家基因的GAPDH的。基因表达被归一化到GAPDH表达，根据2-ΔCT。从相同的排序相同的人口至少有十几个单细胞可以被用作生物复制，以弥补缺乏技术复制。 7。代表性的成果 图1显示了磁共振成像（MRI）扫描的代表与GBM患者。脑肿瘤样本后立即手术，获得病人同意批准的汉密尔顿健康科学/麦克马斯特健康小号ciences研究伦理委员会。每个标本的一部分用于临床常规诊断的神经病理学家。剩余的样品进行处理，如在图2中示出。的肿瘤细胞，一旦镀完整的神经干细胞培养基中，如在图3中示出与生长因子，形式tumorspheres。 表面与神经干细胞标志物CD133和CD15标记的肿瘤细胞中，并通过流式细胞术分析，如在图4中所示。 单细胞不同人群例如 ，CD15 + / CD133 +，CD15 + / CD133-，CD15-/CD133 +，CD15-/CD133-，然后到96孔板（ 图5a）或成AmpliGrid幻灯片（ 图6a）排序。 在96孔板中，单细胞，这些细胞具有自我更新的能力（ 例如，CD133 +细胞），（ 图5b），可以形成tumorspheres（ 图5c）INCUcDNA克隆。可以绘制一个自我更新的图形（ 图5d），计算领域形成每口井的数量。单细胞的排序条件到反应部位的Ampligrid滑动件（ 图6a）。可以从单细胞提取的RNA逆转录使用Advalytix AmpliSpeed（ 图6b）。使用获得的cDNA进行实时RT-PCR（ 图6c）。 <img alt="图1" src="/files/ftp_upload/4111/4111fig1.jpg" /> 图1。磁共振成像（MRI）的病人的GBM。一个月悠久的历史，头痛，呕吐，嗜睡，视力模糊为期一周的历史与一个16岁的女孩的大脑MRI扫描。轴向FLAIR序列显示了一个大的双半球（"蝴蝶"）GBM。 <img alt="图2" src="/files/ftp_upload/4111/4111fig2.jpg" /> 图2。脑肿瘤脑肿瘤样本的处理。要想获取立即手术后病人同意的独立非执行董事。机械分离的，如图所示，（a）和然后酶解法分离通过在每分钟30转的摆动培养箱温育在37℃下为15分钟（二）在脑脊液与Liberase。 <img alt="图3" src="/files/ftp_upload/4111/4111fig3.jpg" /> 图3。 BTIC的人群形成tumorspheres文化。游离脑肿瘤细胞的生长因子与完整的神经干细胞培养基铺在形成tumorspheres。 <img alt="图4" src="/files/ftp_upload/4111/4111fig4.jpg" /> 图4。流式细胞仪分析BTIC人群。（a）远期与侧散射特性提供了一个图像的所有细胞，包括碎片（二）7-AAD的可行性染料染色的细胞从分析中排除。 （三）的统计象限的位置，确定使用相应的同型对照（D）的肿瘤细胞染色，麻面rkers CD15-PE和CD133-APC。 （四）Moflo XDP的细胞分拣机。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4111/4111fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/4111/4111fig5.jpg" /> 图5。限制稀释的BTICs分析显示其自我更新的能力。（一）细胞的不同稀释度进行排序NSC（b）单细胞每孔含200微升到96口井。 （三）经过7天的潜伏期，tumorsphere形成。的二次tumorspheres形态是相同初级tumorspheres的。 （d）本平均x截距值，可以计算出从有限稀释分析，以显示每孔形成的至少一个tumorsphere所需的细胞数量的每个肿瘤亚型。 <img alt="图6" src="/files/ftp_upload/4111/4111fig6.jpg" /> 图6。的一个单一的BTIC细胞的基因表达分析可以使用单细胞RT-PCR技术。（一）单细胞到，可以进行排序从BTIC人口的AmpliGrid幻灯片。 （二）进行cDNA合成对单细胞在AmpliGrid幻灯片使用Advalytix AmpliSpeed​​。 （三）定量实时PCR进行的cDNA合成从单一BTIC细胞。 <table border="1"><tbody><tr><td> 2M的NaCl </td><td> 62毫升 </td></tr><tr><td> 1M氯化钾</td><td> 5毫升</td></tr><tr><td> 1M MgCl 2的 </td><td> 3.2毫升</td></tr><tr><td> 155毫米碳酸氢钠 </td><td> 169毫升</td></tr><tr><td> 1M葡萄糖</td><td> 10毫升</td></tr><tr><td> 108毫米氯化钙 </td><td> 0.9256毫升</td></tr><tr><td> PURELAB超H 2 0 </td><td> 749.84毫升</td></tr></tbody></table> 表1中。人工脑脊液（ACSF） - 1,000毫升。 <table border="1"><tbody><tr><td> 库存基本培养基 </td><td> 500毫升 </td></tr><tr><td> 1:1的DMEM:F12 </td><td> 480毫升</td></tr><tr><td> N2补充</td><td> 5毫升</td></tr><tr><td> 1M HEPES </td><td> 5毫升</td></tr><tr><td>葡萄糖</td><td> 3.0克</td></tr><tr><td> N-乙酰半胱氨酸（60微克/毫升） </td><td> 1毫升</td></tr><tr><td>神经存活因子1（NSF-1） </td><td> 10毫升</td></tr></tbody></table> 表2中。神经干细胞培养基。 NSC完整的媒体 （前取得了新的使用）: NSC基础培养基+ 20 ng / ml的EGF（表皮生长因子）+20 ng / ml的碱性成纤维细胞生长因子（碱性成纤维细胞生长因子）+ 10纳克/毫升LIF（白血病抑制因子）* + 10微升/毫升的抗生素 - 抗真菌剂 *白血病抑制因子（LIF）:此试剂从Millipore包含类细胞因子的白细胞介素6，某otein抑制自发分化的干细胞促进干细胞培养长期的维护。 <table border="1"><tbody><tr><td> 抗体 </td><td> 供应商/ CAT＃ </td><td> 微升/测试（在100μl） </td></tr><tr><td> CD133 / 2 APC（293C3） </td><td>美天旎Biotec/130-090-854 </td><td> 5 </td></tr><tr><td> IgG2b的APC（同种型对照） </td><td>美天旎Biotec/130-092-217 </td><td> 5 </td></tr><tr><td> CD15PE </td><td>贝克曼Coulter/IM1954U </td><td> 5 </td></tr><tr><td> IgG2a的PE（同种型对照） </td><td>贝克曼Coulter/A09141 </td><td> 5 </td></tr><tr><td> 7AAD </td><td>贝克曼Coulter/A07704 </td><td> 10 </td></tr></tbody></table> 表3流量抗体。 <table border="1"><tbody><tr><td> 元件 </td><td> 量（反应） </td><td> 成交量（48反应） </td></tr><tr><td> 2个单细胞RT反应缓冲液</td><td> 0.50微升</td><td> 30.0微升</td></tr><tr><td> RNA酶抑制剂（10 U /μL） </td><td> 0.02μL </td><td> 1.2μL </td></tr><tr><td> 5倍的单细胞RT增强</td><td> 0.15微升</td><td> 9.00微升</td></tr><tr><td>单细胞逆转录酶混合</td><td> 0.04微升</td><td> 2.4微升</td></tr><tr><td> Advablue（OAX04227） </td><td> 0.1μL </td><td> 6.00微升</td></tr><tr><td>无核酸酶的水</td><td> 1微升</td><td>至60微升</td></tr></tbody></table> 表4。单细胞RT-PCR（CAT＃OAX04515）的反转录（RT）混合组成。

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癌干细胞假说10，是根据工作21在白血病，乳腺癌11和脑癌4,5，表明仅有一个相对小的部分的细胞在肿瘤中，称为肿瘤干细胞，具有客户广泛增殖和自我 - 更新。大多数的肿瘤细胞失去增殖的能力和自我更新，因为它们分化成细胞，成为表型的肿瘤签名。找到关键脑肿瘤的人口能够维持肿瘤细胞的脑肿瘤发生的机制和深入了解，使我们能够追溯起源的细胞。 <p cla...

脑肿瘤中最积极和异质性在人类癌症。虽然他们的早期检测和诊断提供了便利的现代神经影像学技术，我们仍然缺乏许多脑肿瘤的根治性治疗方法，特别是对弥漫性，浸润性，或者那些在大脑深处。 脑肿瘤由于他们的性质非常凶悍，经常无法治愈的儿童癌症死亡率的首要原因。胶质母细胞瘤（GBM），在成人中最常见的原发性脑肿瘤，是一种最积极的人类癌症，担心其均匀致命的预后1。高度恶性星形细胞瘤（WHO 4级），通常发生在成人大脑半球，也可以发生在儿童和婴儿。它的增长是快速渗透，诊断的病理特征包括核多形性，微血管增生，坏死2,3。对于adulTS新诊断为GBM，中位生存期很少超出12个月1，所有治疗方法的普遍反应不佳。我们注意到，有许多共享的成体干细胞和癌细胞的功能和遗传相似性，在肿瘤的分子通路，调节大脑正常发育往往是失调的。在应用的研究脑肿瘤干细胞生物学范式，我们是第一个研究人员前瞻性地识别和纯化的人类的GBMS表现出增殖，自我更新的干细胞特性的细胞亚群，4 和体外分化体内 5。我们最初用来描述正常的神经干细胞（NSCs） 体外 6,7到多个儿童和成人脑瘤的培养条件和检测应用，丰富这些干细胞样细胞的细胞分选的神经祖细胞表面标志CD133 ，9。的CD133 +脑肿瘤馏分包含有高得多的频率的肿瘤起始细胞在NOD-SCID小鼠的大脑5,10比CD133-馏分。这正式成立，只有一种罕见的脑肿瘤细胞与干细胞特性的子集是肿瘤起始，赢得他们的名称为"脑肿瘤启动细胞"或"BTICs"。新的识别BTICs到人脑肿瘤提供了新的见解，给予大力支持的癌症干细胞假说10-13许多实体肿瘤的基础，并建立了一个新的细胞目标的更有效的癌症疗法14-20。重点杀死大量的肿瘤的治疗，可能会错过难得的干细胞样的分数，使肿瘤继续增长。重点杀死癌症干细胞的治疗，可能为脑肿瘤患者提供更好的治疗和预后。 在为了研究BTIC的人群，我们有完善我们的丘尔重新协议，专门选择在人类脑肿瘤的细胞群具有干细胞的特性。无血清，神经干细胞（NS​​C）媒介允许未分化的干细胞状态的维持，和此外，碱性成纤维细胞生长因子（bFGF），表皮生长因子（EGF），白血病抑制因子（LIF），允许多有力，自我更新，和可扩展的人类tumorspheres增殖。在这里，我们描述的原发性脑肿瘤的处理和培养NSC媒体，以丰富BTIC人口参与的方法， 我们都要求我们的实验模型系统"BTIC的的病人分离"强调一个事实，这些都只是最低限度的条件下培养干细胞选择的干细胞群的细胞条件。随后的关键干细胞标志物，如CD133和CD15和流式细胞仪分析免疫标记的BTIC人口中也有描述。然后，我们讨论有限稀释法，这有助于研究自我更新的BTICs潜力。最后，我们将探讨这些稀有细胞的基因表达分析的，分拣单细胞上AmpliGrid幻灯片和执行单细胞RT-PCR。这些技术也适用于其他脑肿瘤如髓母细胞瘤，室管膜瘤和儿科神经胶质瘤。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 1:1的DMEM:F12 </td><td> Invitrogen公司</td><td> 11320-082 </td></tr><tr><td> N2补充</td><td> Invitrogen公司</td><td> 17502-048 </td></tr><tr><td> 1M HEPES </td><td>欧洲野牛</td><td> 330-050-EL </td></tr><tr><td>葡萄糖</td><td> Invitrogen公司</td><td> 15023-021 </td></tr><tr><td> N-乙酰半胱氨酸</td><td> Sigma Aldrich公司</td><td> A9165-25G </td></tr><tr><td>神经的生存因子-1（NSF-1） </td><td>龙沙Clonetics </td><td> CC-4323 </td></tr><tr><td>表皮生长因子（EGF） </td><td> Sigma Aldrich公司</td><td> E9644 </td></tr><tr><td>碱性成纤维细胞生长因子（bFGF） </td><td> Invitrogen公司</td><td> PHG0261 </td></tr><tr><td>白血病抑制因子（LIF） </td><td>密理博</td><td> LIF1010 </td></tr><tr><td>抗生素/霉菌性</td><td>欧洲野牛</td><td> 450-115-EL </td></tr><tr><td> Liberase TM </td><td>罗氏公司</td><td> 05 401 119 001 </td></tr><tr><td>氯化铵溶液</td><td>干细胞技术</td><td> 07850 </td></tr></tbody></table>

processing of primary brain tumor tissue for stem cell assays and flow sorting

脑肿瘤通常是由不同的形态的细胞，表达出各种神经谱系标记。只有小部分的肿瘤细胞与干细胞的特性，称为脑肿瘤启动细胞（BTICs），具备的能力以及多种细胞类型分化，自我更新，并启动体内的肿瘤。我们采用原本用于正常的神经干细胞（NS​​Cs）的各种人脑肿瘤，结果发现，这种文化的方法，具体选择干细胞样的人群的文化条件。无血清培养基（NSC）允许的未分化的干细胞状态的维持，和碱性成纤维细胞生长因子和表皮生长因子的添加允许多强有力的，自我更新，增殖和膨胀tumorspheres。 为了进一步刻画每个肿瘤的BTIC人口，我们通过流式细胞仪检测细胞表面标志物进行评估。我们也可以更具体的characteriz关注的群体排序BULLETIN。自我更新检测到96孔板上执行单BTICs排序条件的形成在37温育后的tumorspheres°C表示的干细胞或祖细胞的存在。某一特定人群的多个细胞的数量也可以在不同的井进行排序，有限稀释分析，分析的自我更新能力。我们也可以研究在一个特定的细胞群的差异表达基因的单细胞RT-PCR。 以下协议描述了我们的程序主人体标本的分离和培养，以丰富的BTIC人口，以及分离的tumorspheres。此外，还包括用于染色，流式细胞仪分析或排序，自我更新的检测，和单细胞RT-PCR方法的协议。

Watch this Scientific Journal Video about Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting at JoVE.com

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

人脑肿瘤发病机制，鉴定的脑瘤开始的细胞（BTICs），罕见的一个异质性肿瘤具有干细胞特性的细胞内，提供了新的见解。我们已调整了特定的文化条件，以丰富BTICs，我们经常使用流式细胞仪检测，以进一步丰富这些人群。对这些孤立的细胞自我更新的检测和RT-PCR单细胞的转录分析可以随后进行。

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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18.0K 次观看.  McMaster University. 人脑肿瘤发病机制，鉴定的脑瘤开始的细胞（BTICs），罕见的一个异质性肿瘤具有干细胞特性的细胞内，提供了新的见解。我们已调整了特定的文化条件，以丰富BTICs，我们经常使用流式细胞仪检测，以进一步丰富这些人群。对这些孤立的细胞自我更新的检测和RT-PCR单细胞的转录分析可以随后进行。

视频: Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

制备髓源性抑制细胞从幼稚和胰腺癌荷瘤小鼠，用流式细胞仪和自动磁性细胞分选（AutoMACS）（MDSC）

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

科研

医学

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

加工的主要脑肿瘤组织的干细胞检测和流量排序

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