我们承认USF流式细胞仪核心设施。我们想感谢丹尼斯库珀博士资源共享。我们也想感谢他们的协助玛雅科恩，劳拉·彭德尔顿和戴安娜拉图尔建立和拍摄这部影片。神经网络的支持NSF FG-LSAMP桥博士奖学金人力资源开发＃0929435。这项工作是由美国癌症学会院校研究资助＃93-032-13/Moffitt癌症中心颁发到TG。

开始之前，请准备以下解决方案： 3％，染色媒体（SM）： -3％的胎牛血清（FBS），1X的磷酸盐缓冲液（PBS） 磁珠缓存（MB）： - 牛血清白蛋白（BSA）的0.5％白蛋白在1XPBS 1。收获从小鼠脾脏<ol><li>皮下注入1.5×10 5小鼠Panc02细胞悬浮在100μL1X PBS 6-8周岁的C57BL / 6小鼠（哈伦）（荷瘤;结核病）。对照组（幼稚）收到100μL1XPBS。 </li><li>注射后约4周，安乐死小鼠二氧化碳窒息。 </li><li>收获从小鼠脾脏钝性使用镊子和剪刀剥离，然后重使用平衡。脾脏在不同的地方，打成50毫升含1×PBS的锥形管。 </li></ol> 2。生成一个单细胞Suspensio的n的脾脏白细胞 所有程序应在无菌环境下的生物安全罩及细胞和抗体放在冰上。 <ol><li>组装插入朝下方杯开幕丝网细胞分离筛。然后，扣环插入螺纹面积的开槽端和使用环的关键，拧紧固定环，从而控股到位的屏幕。将聚集在一个Petri菜含有10毫升1×PBS筛。 </li><li>池脾脏和使用玻璃杵研磨脾脏对细胞分离筛的筛网进入培养皿。各治疗组小鼠的重复。 </li><li>到50毫升锥形管使用70微米的细胞过滤器和5毫升血清移液器过滤细胞悬液。 12,000 RPM（250-300 XG）离心5分钟。 </li><li>取出5毫升1×每脾红细胞裂解液上清，重悬沉淀。吸取积极向上和向下。在室温下孵育5分钟。停止反应，加入20毫升1×PBS。吸取积极向上和向下。 12,000 RPM（250-300 XG）离心5分钟。 </li><li>取出20毫升无菌1×PBS上清，重悬沉淀。吸取积极向上和向下。 </li><li> 50μL，相当于5X10 5-1×10 6细胞（1×10 7细胞/ ml - 2×10 7细胞/ ml）计数细胞，用台盼蓝和血球和所需的浓度在3％的SM重悬。 </li></ol> 3。细胞表面染色/使用流式细胞仪免疫表型的MDSC <ol><li> 96 V为控制和补偿控制的实验样品和单污渍底板标注的井（没有污点，生理盐水;的Mac-1-FITC; GR-1装甲运兵车和DAPI）。 </li><li>添加5X10 5-1×10 6细胞，相当于50 /脾到各自的井井液在96孔V型底板。离心分离板在5分钟转速12,000 rpm（250-300 XG）。 </li><li>准备“预混”鼠标BD FC块在3％的SM 1.5毫升离心管上的冰，稀释（大鼠抗小鼠CD16/32单克隆抗体）（MM）。作为一个起点，最终体积为50μL使用1微克，在3％的SM FC块，每口井。 </li><li>仔细取出上清液从每个井的96-V型底板的快速反相板和背面，不破坏细胞颗粒在一个废物容器或水槽。 </li><li>涡，短暂离心5秒FC块MM和添加50μL，在96孔V型底板所有颗粒。拌匀，轻轻吹打下来，留在他们的水井样品。孵育15分钟，在黑暗冰板。离心5分钟转速12,000 rpm（250-300 XG）板。 </li><li>准备“师父混合”（MM）的3％，在冰上的1.5ml离心管中的SM稀释的荧光标记抗体，而样品与FC块孵化。抗体应该被滴定染色程序，以确定最佳的稀释。作为一个起点，在3％的SM结合的Mac的1-FITC 1:25稀释和1:20稀释的GR-1-APC最终体积为50μL，每个样品以及。 </li><li>从每个井的96-V型底部小心取出上清液，作为先前所描述的。 </li><li>旋涡，短暂离心5秒共轭荧光染色抗体的MM，添加50μL，对照组和实验颗粒。轻轻吹打上下拌匀。对于单染色的赔偿，添加的Mac的1-FITC 1:25稀释，1:20稀释的GR-1-APC和75毫微克/毫升的DAPI，最终体积为50μL每口井的3％的SM各自的水井。加入50微升3％的SM不染井（无污点）。拌匀孵育细胞在96孔V型底板块在黑暗的冰上30分钟。 </li><li>标签流式细胞管（5毫升，12毫米×75毫米的聚苯乙烯圆底管）对应到每口井在96孔V型底部的板块。加入200μl3％的SM每个流式细胞仪管。 </li><li>离心转速12,000 rpm（250-300 XG）板为5分钟，去除上清。洗净加入100微升3％的SM每个颗粒颗粒混合，轻轻吹打上下。离心5分钟转速12,000 rpm（250-300 XG）板。重复洗涤步骤一次。 </li><li>从每个井的96-V型底部小心取出上清液，作为先前所描述的。每个颗粒和悬浮在100微升3％的SM，拌匀。 </li><li>转让的96-V型底板，以及从每100μL悬浮颗粒，他们分别标记的流式细胞仪管。 </li><li>流式细胞仪分析之前，添加75毫微克/毫升的DAPI控制和实验样品和单一的DAPI染色补偿控制。 </li><li>执行流式细胞仪数据采集的MDSC百分比。使用负（没有的污渍控制）和单阳性对照执行赔偿。设立这样一个点的情节，显示日志规模远期（FSC）与侧散射（SSC），白细胞人口可确定利益。画一个大的门，但不包括所有白细胞最低的前部和侧分散的碎片和团块。从这个父门，创建一个新的散点图显示南南合作的DAPI（直播）细胞与DAPI和门。选择这个新门的人口，并创建一个点图上显示您的双阳性的Mac-1与GR-1和门（MAC-1 + GR-1 +）的MDSC。 </li></ol> 4。 GR-1 +白细胞磁富集<ol><li>等分1×10 7到适当标记的流式细胞仪管和离心机在12000转5分钟（250-300 XG）剩余未染色白细胞。 </li><li> GR-1-PE抗体的MM准备在1.5 ml离心管。高达10 7细胞，用50μLMB的一个GR-1-PE抗体1:10稀释，每个样品。对于更大的手机号码，相应扩展卷。短暂离心5秒钟，流式细胞仪管中的白细胞和孵育15分钟，在4°C黑暗环境中。 </李&gt; <li>加入2毫升的MB流式细胞仪管，离心5分钟转速12,000 rpm（250-300 XG），弃上清。 </li><li>准备在1.5 ml离心管的反体育微珠MM。高达10 7细胞，用200μLMB的1:4稀释的反PE微珠，每个样品。对于更大的手机号码，相应扩展卷。短暂离心5秒钟，流式细胞仪管中的白细胞和孵育15分钟，在4°C黑暗环境中。 </li><li>加入2毫升的MB流式细胞仪管，离心5分钟转速12,000 rpm（250-300 XG），弃上清。重悬浮颗粒在3毫升的SB。通过70微米滤网过滤到一个新的，标有50毫升锥形管。 </li><li>准备和总理汽车磁珠临分离。所有笔芯瓶，如果有必要与适当的解决方案和空废液瓶。打开仪器检查液容器和初始化后列（S）的状态。所有的符号应该是绿色的。在菜单上，选择上“分离”其次是菜单栏“洗”从较低的菜单栏。选择“冲洗”，从弹出的选项“运行”开始启动过程。一旦启动过程成功完成后，仪器将显示，这是“分离”状态“菜单下。 </li><li>馏分收集和B行负在行C.积极馏分收集的15毫升锥形管排，50毫升锥形管的磁标记细胞选择适合管的尺寸和地方的50毫升锥形管冷冻试管架</li><li>选择“POSSEL_S”积极选择敏感的模式，从样品中标记的靶细胞的细胞分离方案。磁标记的靶细胞被保留在自动贩卖机列;未标记的细胞释放到负的馏分收集管行B.在磁铁自动回缩，将标记的靶细胞释放到积极的收集管，行管ç机架。 </li></ol>+浓缩白细胞后排序分析<ol><li>验票GR-1 +和Gr-1 -使用台盼蓝和血球的分数。重悬细胞所需的浓度在3％的染色介质，使50μL相当于5X10 5-1×10 6细胞（1×10 7细胞/ ml - 2×10 7细胞/ ml）和50μL转移到相应标记的流式细胞仪管。 </li><li>准备仅适用于Mac-1的MM，在3％的SM终体积为50μL样品1:25稀释。染色细胞和单GR-PE，MAC-1 FITC和DAPI染色赔偿准备流式细胞仪分析。加入200微升3％，SM和DAPI可行性染料如前所述。 </li><li>执行流流式细胞仪的数据采集，以确定GR-1 +和Gr-1 -比例也比较的MDSC百分比前和后autoMACS的富集。 </li></ol> 6。代表结果在这里，我们显示ŕ<html> GR-1 +汇集，天真的MDSC（ 图1）排序随后流式细胞仪脾脏白细胞富集autoMACS之代表结果。幼稚白细胞1×10 6的Mac-1-FITC和GR-1-APC抗体染色，以确定使用的BD LSRII仪器的MDSC百分比，前到autoMACS排序。幼稚白细胞1×10 7，然后染色，抗GR-1-PE和PE的微珠富集GR-1 +使用一个autoMACS临分离的白细胞抗体。后autoMACS富集，GR-1的百分比GR-1 +和Gr-1 -收集馏分使用流式细胞仪进行了评价。 1×10 6 GR-1 +白细胞被拆除的Mac-1-FITC抗体进行分析和比较丰富的MDSC百分比，汇集非丰富，汇集通过流式细胞仪检测荷瘤白细胞的幼稚白细胞染色。 fig1.jpg“/&gt; 图1。朴素GR-1 +排序的MDSC流式细胞仪白细胞AutoMACS富集。胰腺癌荷瘤和天真的小鼠脾脏收获并加工成单细胞悬液。幼稚白细胞表面流与Mac-1和GR-1荧光标记的抗体染色流式细胞仪分析，autoMACS富集（一）前。流流式细胞仪分析GR-1 +（乙）和GR-1 - （）分数后autoMACS GR-1 +细胞GR-1-PE抗体和抗PE的微珠染色汇集天真leuckocytes的富集。流仪分析的MDSC和Gr-1 +百分比后GR-1-autoMACS富集汇集幼稚白细胞（四）+细胞相比，非浓缩汇集来自荷瘤小鼠的白细胞（五）（天真的小鼠，每组5荷瘤小鼠，N = 3）。门代表的等高线图和直方图的MDSC和GR-1的百分比。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+responses+of+human+regulatory+T+cells+(Treg)+and+effector+T+cells+to+rapamycin.">Differential responses of human regulatory T cells (Treg) and effector T cells to rapamycin.</a> PLoS One. 4, e5994 (2009).</li></ol>

没有利益冲突的声明。

这是一个详细的方法处理和immunophentyping的MDSC人口，是适用于各种动物模型不同的淋巴组织。特别，autoMACS富集，可用于各种白细胞的人口，包括GR-1枯竭脾4，从脾和淋巴结肿大5粒细胞亚群的净化，分 ​​离骨髓中性粒细胞14和纯化的CD8 + T细胞从脾隔离和淋巴结15。无论所需的淋巴组织产生白细胞的单细胞悬液的细胞群体的利益，需要最佳表面染色，流式?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> 1X磷酸盐缓冲液</td><td>热科学胎牛血清</td><td> SH30028.02 </td><td>的Ca 2 + /镁2 + /苯酚红</td></tr><tr><td>白蛋白牛血清白蛋白（BSA） </td><td> Sigma-Aldrich公司</td><td> A7906 </td><td>让牛血清白蛋白溶解在PBS原状;无菌环境</td></tr><tr><td>胎牛血清（FBS）， </td><td>热科学胎牛血清</td><td> SV3001403HI </td><td>热灭活;无菌环境</td></tr><tr><td>大鼠抗小鼠单克隆抗体CD16/32（FC块） </td><td> BD公司</td><td> 553142 </td><td>无菌环境</td></tr><tr><td>抗鼠细胞CD11b（MAC-1）FITC标记</td><td> eBiosciences </td><td> 11-0112 </td><td>不育环境</td></tr><tr><td>抗鼠Ly6G（GR-1）APC </td><td> eBiosciences </td><td> 17-5931 </td><td>无菌环境</td></tr><tr><td>抗鼠Ly6G（GR-1）PE </td><td> eBiosciences </td><td> 12-5931 </td><td>无菌环境</td></tr><tr><td>的DAPI </td><td> Invitrogen公司</td><td> D1306 </td><td>连续稀释无菌环境</td></tr><tr><td>细胞分离筛</td><td> Sigma-Aldrich公司</td><td> CD1-1KT </td><td>使用高压灭菌前</td></tr><tr><td> 70微米过滤器</td><td> BD公司</td><td> 352350 </td><td>无菌环境</td></tr><tr><td> 1X红细胞裂解液</td><td> eBiosciences </td><td> 00-4333-57 </td><td>温暖到室温后使用无菌环境</td></tr><tr><td>培养皿</td><td> Fisher Scientific则</td><td> 08-757-12 </td><td>无菌环境</td></tr><tr><td> 50毫升CONICAL管</td><td> Thermo Scientific的</td><td> 339652 </td><td>无菌环境</td></tr><tr><td> 5毫升12X75mm聚苯乙烯圆底管</td><td> BD公司</td><td> 352054 </td><td>无菌环境已知流式细胞仪管; </td></tr><tr><td> 96-V型底板</td><td>康宁</td><td> 3897 </td><td>无菌环境</td></tr><tr><td>台盼蓝</td><td> cellgro </td><td> 25-900-CI </td><td>无菌环境</td></tr><tr><td>体育微珠</td><td>美天旎生物技术</td><td> 130-048-801 </td><td>无菌环境</td></tr><tr><td> AutoMACS临分离</td><td>美天旎生物技术</td><td> 130-092-545 </td><td></td></tr><tr><td> AutoMACS列</td><td>美天旎生物技术</td><td> 130-021-101 </td><td></td></tr><tr><td> AutoMACS运行缓冲液</td><td>美天旎生物技术</td><td> 130-091-221 </td><td&gt; </td></tr></tbody></table>

preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)

的MDSC是一个不成熟的巨噬细胞，树突状细胞和粒细胞积聚在病理条件下的淋巴器官，包括寄生虫感染，炎症，创伤后应激，移植物抗宿主病，糖尿病和癌症1-7异构人口。在小鼠的MDSC明示的Mac-1（细胞CD11b）和GR-1（Ly6G和Ly6C）表面抗原。重要的是要注意的MDSC荷，它们显着扩大在各个主机研究和压制7-10天真对口相比抗肿瘤免疫反应。然而，根据病理状态，有不同的MDSC亚与不同的机制和抑制11,12的目标。因此，有效的方法来隔离可行的MDSC人口是重要的，在阐明各自不同的分子机制抑制在体外和体内 。 近日，在Ghansah组报告的MDSC在小鼠胰腺癌模型的扩展。我们的肿瘤轴承的MDSC显示动态平衡的损失，并增加抑制功能比较幼稚的MDSC 13。的MDSC的百分比显着天真与荷瘤小鼠的淋巴车厢。这是一个重大的警告，这往往阻碍了这些的MDSC准确的比较分析。因此，丰富天真小鼠GR-1 +白细胞荧光激活细胞分选（FACS）前提高纯度，活力和排序的时间显着降低。然而，丰富的GR-1 +荷瘤小鼠的白细胞是可选的，因为这些丰富的排序快速流式细胞仪。因此，在这个协议中，我们描述了一个高效的方法，免疫的MDSC从排序的MDSC及时天真小鼠脾脏和丰富的Gr-1 +白细胞。免疫C57BL / 6小鼠接种小鼠Panc02 CE的LLS皮下而天真的小鼠接受1XPBS。大约30天接种后脾脏被采伐和加工成单细胞悬液采用细胞分离筛。脾然后红细胞（RBC）的裂解，这些白细胞等分对MAC-1和GR-1免疫的MDSC百分比用流式细胞仪使用荧光标记抗体染色。在一个类似的实验，从整个天真的小鼠白细胞染色与荧光标记的GR-1抗体，培养与PE的微珠和正选择使用一个自动化的磁性活化细胞分选（autoMACS）分离。接下来，GR-1 +白细胞等份与Mac-1抗体染色，以确定在使用流式细胞仪的MDSC百分比增加。现在，这些GR1 +丰富的白细胞是准备用于比较分析（天真与荷瘤） 在体内和体外的MDSC排序流式细胞仪</em&gt;的检测。

Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

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

这是一个迅速和全面的免疫髓源性抑制细胞（MDSC）和丰富GR-1的方法<sup&gt; -</sup&gt;从小鼠脾脏白细胞。这种方法使用流式细胞仪和AutoMACS细胞排序丰富可行的GR-1<sup&gt; -</sup&gt;白细胞之前使用的MDSC排序，以流式细胞仪<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;检测。

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 ...

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JoVE Journal

Medicine

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

26.1K 次观看.  University of South Florida Morsani College of Medicine. 这是一个迅速和全面的免疫髓源性抑制细胞（MDSC）和丰富GR-1的方法 -从小鼠脾脏白细胞。这种方法使用流式细胞仪和AutoMACS细胞排序丰富可行的GR-1 -白细胞之前使用的MDSC排序，以流式细胞仪在体内和在体外检测。

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

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

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

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 ...

科研

医学

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 ...

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 ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

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 ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

纳米抗体和基于抗体的验证<em&gt;在体内</em&gt;肿瘤异种移植NIRF成像的小鼠实验使用<em&gt;离体</em&gt;流式细胞仪和显微镜

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

分子探针的优化，以确定细胞死亡的光合生物（<em&gt;铜绿微囊藻</em&gt;）使用流式细胞仪

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.