We thank Ms. Danae Sharp and Ms. Sheena Daignault for technical assistance. We thank Dr. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Dr. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, for providing cell lines, the Imaging and Flow Cytometry Facility at the Centenary Institute for outstanding technical support. We thank Mr. Chris Johnson and Dr. Andrew Barlow for Volocity software technical support. N.K.H. is a Cameron fellow of the Melanoma and Skin Cancer Research Institute, Australia. K.A.B. is a fellow of the Cancer Institute New South Wales (13/ECF/1-39). W.W. is a fellow of the Cancer Institute New South Wales (11/CDF/3-39). This work was supported by project grants RG 09-08 and RG 13-06 (Cancer Council New South Wales), 570778 and 1051996 (Priority-driven collaborative cancer research scheme/Cancer Australia/Cure Cancer Australia Foundation), 08/RFG/1-27 (Cancer Institute New South Wales), and APP1003637 and APP1084893 (National Health and Medical Research Council).

1. FUCCI转导和细胞培养<ol><li> FUCCI转导<ol><li>创建细胞系稳定表达FUCCI使用慢病毒共转导如前所述7构造mKO2-hCdt1（30-120）和MAG-hGem（1-100）15。 注：FUCCI系统现在市售的。 </li><li>生成子的克隆与明亮的荧光由单细胞分选。排序单阳性细胞都AG和KO（黄色）通过荧光激活细胞分选入96孔板如前所述7,16。 </li></ol></li><li>黑色素细胞培植<ol><li>培养C8161人黑素瘤细胞如前所述7,17。 </li></ol></li></ol> 2.三维球体的形成（如前所述3,9） <ol><li>琼脂糖板的准备<ol><li>溶解1.5％琼脂糖（或稀有琼脂）在100毫升的Hank氏平衡盐溶液（HBSS）或磷酸盐buffereð盐水（PBS）通过在微波中3-5分钟旋流沸腾。确保琼脂糖完全溶解。 </li><li>立即用多通道移液器分配每孔100μl的琼脂糖溶液至平底96孔组织培养板。 注意：琼脂糖可以以提示固化后的短时间内，为了克服这个问题，必须提示板之间改变，如果使多个琼脂糖平板。 </li><li>留在平坦表面上96孔板硬化琼脂糖至少1小时。 </li></ol></li><li>电池的制备和覆盖到琼脂糖<ol><li>生长细胞到约80％汇合的T75烧瓶。用10毫升HBSS清洗。 </li><li>用1.5毫升的0.05％胰蛋白酶/乙二胺四乙酸（EDTA），重悬在10ml正常培养基分离细胞。 </li><li>计数使用血球18或其他自动计数法的细胞。 </li><li>重悬的细胞以在范数25,000个细胞/ ml的终浓度人网上平台。 </li><li>覆盖琼脂糖孔用200μl细胞悬浮液为每孔5,000个细胞的终数目。 </li><li>回位盘到细胞培养孵化器（5％CO 2和37℃），以形成球状体在约3天。 注意：采取以形成球体的时间不同的细胞系之间变化。某些细胞系不形成使用该方法在所有的球状体。 </li><li>图像球体形态与使用4X物镜和相衬滤波器倒置显微镜。的细胞系，成功地形成球状体将产生紧凑，大致球状细胞聚集，并且应该有每口井只有一个球体。 注意：可选：一旦球体形成，它们可以被移植到胶原或其他基质的生长及侵袭3,7,17 ​​的进一步分析。从胶原除去球体，治疗用500μl的2毫克/毫升胶原酶在37℃下，直至胶原溶解足够的spher的OID来自由入培养基（约30分钟）。轻轻地取出用1毫升吸管球体。 </li><li>用于切片/成像修复球状体（无论是分离的由胶原或生长3-5天在琼脂糖）在10％的中性缓冲的福尔马林（小心：福尔马林应在通风橱中使用）至少2小时，在室温（RT） ，或隔夜（O / N）在4℃。球状​​体可以被存储在HBSS在4℃下数天。 </li></ol></li></ol> 3.球体Vibratome切片<ol><li>溶解5克在100ml的HBSS低熔点琼脂糖在500毫升玻璃瓶中微波炉。使用带有旋转介质热定型。注意：保持盖子松动时沸腾的溶液中，使得该压力可以被释放。使用防护手套，防热处理热玻璃奶瓶时要保护手烫伤。 </li><li>等到琼脂糖稍微冷却 - 这样的瓶子可以不烧手触摸。 </li><li>倒approximately 2毫升琼脂糖成Coulter计数器杯或类似塑料模具的底部。立即吸固定球体（见2.2.8）中的液体可能的最小体积使用1ml吸管。加入球体的琼脂糖。几个球体（最多10个）可以每杯加入。 </li><li>覆盖球体用2毫升更多的琼脂糖。为此快，从而避免琼脂糖硬化的第二层中加入之前。 </li><li>允许琼脂糖在RT在暗处硬化，至少3小时。在这一点上的杯子可被存储的时间很短，在4℃用2-3毫升的HBSS覆盖，以防止脱水。 </li><li>从杯子琼脂糖删除。修剪含有球状体，以便它装配在vibratome金属块，和球状体接近琼脂糖顶部的琼脂糖。球状​​体可以被看作是在琼脂糖内小白物体。强力胶琼脂糖块。 </li><li>装满蒸馏水vibratome并安装在vibratome块。摆好vibratome削减100微米的部分使用低转速（设定2）和高振动（设置8）。设置切刃角至16°。转vibratome上，打开vibratome光并切断从琼脂糖的顶部部分直到100微米的球体部分被切断。将切球体部分为HBSS的24孔板。 </li><li>选择中间部分的图像分析。山路段上的幻灯片被转移部分平板上使用镊子滑动。填有10毫升注射器筒与真空油脂;为了防止在安装介质从流失滑动的边缘，并且帮助密封盖玻片下的部分添加的细线的周围部的边缘真空润滑脂。覆盖部分与封固剂和盖玻片。密封盖玻片指甲油的边缘。幻灯片保存在4℃前成像。 </li></ol> 4.共聚焦图像采集<ol><li>走中间FUCCI球体部分的使用10X的Z切片图像或20X物镜如前所述7。确保没有重叠或串扰Kusabira Orange2和Azami绿色之间发生。如果比较多个球体部分的图像分析，保持激光功率和其他设置的样品之间的相同。 </li></ol> 5.赫斯特染料扩散分析的流量排序<ol><li>转移活球状体（琼脂糖播种后3-5天），以15毫升管。让球体重力沉降到管底部。几个球体可以每管被染色。需要大约20球状体来获得足够的细胞数量用于流式细胞术。 </li><li>删除多余的介质，并加入1毫升10μM赫斯特稀释在普通培养基上。孵育球体在37℃下约1小时。用温柔轻拂孵化过程中一次或两次，悬浮球体。 注意：培养时间可以变化，以改变多远的Hoechst染料渗入球体。这将有所不同的基础上球体的尺寸，密度和细胞系。对于一些大型/致密的球体可以有最大的染料渗透小于100％。 </li><li>用5毫升的HBSS利用重力沉降轻轻冲洗球体。 <ol><li>可选：对于染料渗透成像：在室温或O / N固定球体用10％中性缓冲福尔马林至少2小时，在4℃。制备vibratome部分，如在步骤3和4如上所述安装在滑动和图像上的共焦。 </li></ol></li><li>至在37℃下制备的细胞进行流式细胞术，trypsinize球状体用1ml 0.05％胰蛋白酶/ EDTA约30分钟，轻弹每10分钟，打破球体分开。 注意：球状体是很难得到单细胞悬液，条件可能需要优化。这一过程后C8161 4日龄的球状体生存能力约95％。 </li><li>染色细胞根据制造商的协议的近红外活/死染色。用HBSS洗涤，然后用1修复毫升10％中性福尔马林缓冲液大约30分钟。细胞可被存储在HBSS在4℃下进行流分析前几天。 </li></ol>赫斯特彩绘FUCCI球体的6流式细胞仪分析<ol><li>确保单元，不以通过细胞过滤网来成群在一起。重悬的Hoechst以1-5×10 6个细胞/ ml的浓度染色FUCCI球状体在冰冷的FACS洗涤（PBS中的5％胎牛血清）。转移200μl的样品，以5毫升圆底管中。 </li><li>准备以下单一颜色对照样品中6.1描述：未染色的黑色素细胞;贴壁黑色素细胞染色用10微米赫斯特1小时; Azami绿色只表达黑色素瘤细胞;和Kusabira橙色仅表达黑色素瘤细胞。 注：单色控制可固定在福尔马林中并存储在FACS有0.1％的叠氮化钠洗在4℃数周甚至数月。 </li><li>运行单细胞悬液Ø呐流式细胞分析仪适当的激光和单色/未染色的控制如前所述7,16。 </li><li>使用商业软件，流式细胞仪等的FlowJo如下分析内部与外部球体细胞： <ol><li>通过门控的主要细胞群的前向散射光（FSC）与侧散射光（SSC）清除杂物。 </li><li>通过门控使用FSC（区）与FSC（高）的单细胞移除双峰</li><li>门的活细胞通过门控上的活/死了较低的人口。 </li><li>限定Hoechst的高细胞，根据来自用Hoechst贴壁细胞的阳性信号选通，为“外”的细胞。 </li><li>定义Hoechst的低或阴性细胞，根据来自未染色的控制信号选通，为“内部”的细胞。 </li><li>选通内外种群后，细胞可进一步选通为FUCCI红色（G1），黄色（S早期），绿（S / G2 / M）或负（早期G1）。 注：C可能需要使用单一的色彩控制ompensation正确定义FUCCI红色，黄色，绿色和阴性群。 </li></ol></li><li>一旦测定了优化和通过共焦显微镜Hoechst的渗透验证，而不固定在流动细胞分选，如前所述7,16为活的细胞亚群的进一步分析运行的细胞。 </li></ol> FUCCI球体部分7.图像分析<ol><li>打开软件如 。 Volocity，选择定量唯一的配置。 </li><li>创建新库。从共聚焦拖动原始数据文件到库导入FUCCI球体共聚焦图像文件到软件中。或者，使用文件&gt;导入命令。 </li><li>去测量标签来建立一个图像分析协议。该协议是通过拖放以正确的顺序相应的命令在协议如下窗口建设。 </li><li>查找OBJ学分的绿色通道：拖动找对象命令（在&#39;查找&#39;）复制到协议窗口中，选择正确的通道，在找对象protocol.Add一个开放的命令（在&#39;加工&#39;中），然后一个单独的触摸物体命令（在“加工”发现 - 这将分离细胞）。排除由粒径&lt;50微米的对象（在“过滤”发现 - 这将排除小非细胞对象）。 注意：变量如查找对象的阈值，开放迭代次数，对象的大小要分离和对象的大小，以排除必须手动优化，直到对象掩码匹配图像。 </li><li>发现在红色通道对象：重复如上的红色通道找对象。绿色和红色协议可能需要单独的调整。 注：由于细胞核G2是，绿核往往稍大。 </li><li>确认发现是正确的对象：确认红色和绿色物体匹配红色和绿色的核的图像中通过关闭和绿色和红色通道（使用隐藏或显示信道命令），同时显示由所述协议发现红色和绿色口罩。反馈选项（尺寸&gt;反馈选项）可以用来修改对象口罩的外观。 </li><li>查找黄色的物体（早期S期细胞）：要找到黄色的单元格，添加一个相交红配绿细胞命令（在“合并”中）。排除按大小的物体（&lt;50微米，在“过滤”中）排除任何非小细胞对象。 </li><li>查找独家红色物体（G1期细胞）：要找到专门红核，减黄细胞的红细胞（在“合并”中）。按尺寸&lt;50微米，（在&#39;过滤&#39;中）排除对象删除创建的任何非小细胞对象。 </li><li>查找专门绿色物体（S / G2 / M期细胞）：重复的绿色通道找到专门的绿色。 没有德：如果仍有剩余的非细胞的目的，一个滤波器人口命令可以加入到异绿色或红色专属的协议（在“筛选”中）仅保留对象在具有形状因子大于0.25。这将删除非圆形物体。 </li><li>找到球体外形：查找使用查找球体轮廓对象命令中的绿色或红色通道（无论有更多的细胞周围的球体边缘）（在“查找”中）。甲低得多的阈值（在查找中找到的对象的变量）将需要被用来找到球体轮廓相比，个别细胞。 <ol><li>使用关闭命令加入对象（在&#39;加工&#39;中），然后填写对象孔（在&#39;加工&#39;中），然后用细过滤器（在“过滤”中）从对象去除噪声。最后，通过尺寸排除对象以除去较小的物体&lt;30000微米（根据不同的旋转椭球体的大小）。 注意：此协议将需要手动优化，直到球体轮廓是准确的。甲明图像，也可以使用 - 但是这通常是不太准确与球体部分，和一个反相命令（在&#39;结合&#39;找到）将需要被使用。 </li></ol></li><li>测量核的距离以球体外形：测量距离从黄色，完全绿色和红色的独家人口从小区重心的球体outline.To可视化的最小距离的边缘（在“有关&#39;中），这应该是从小区到最近的球体边缘，打开在反馈选项（尺寸&gt;反馈选项&gt;关系）的关系选项卡显示的距离。 </li><li>保存的协议。该协议可以被重新应用于使用测量其他图像&gt;恢复协议命令。 </li><li>创建一个测量项目（测量&gt;进行测量项目）。数据可以使用分析来分析功能。 <ol><li>转到在测量项目的分析选项卡。转到分析菜单（分析&gt;分析），分析从细胞质心（红色，绿色或黄色）的球体边缘，总结数量和人口组织的最小距离。 </li><li>计数在距边缘有一定距离中发现的细胞的数目创建一个过滤器（分析&gt;过滤器）。的过滤器在图2中最小距离是根据Hoechst的渗透（例如最小距离小于80给出了“外”人口）。另外，原始数据导出到电子表格进行进一步的分析。 </li></ol></li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+tumour+heterogeneity+in+melanoma+therapy:+how+do+we+address+this+in+a+novel+model+system?.">Dynamic tumour heterogeneity in melanoma therapy: how do we address this in a novel model system?.</a> Melanoma Manag. 2, 93-95 (2015).</li><li>Desoize, B., Jardillier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+resistance:+a+paradigm+for+clinical+resistance.">Multicellular resistance: a paradigm for clinical resistance.</a> Critical reviews in oncology/hematology. 36, 193-207 (2000).</li><li>Sakaue-Sawano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+spatiotemporal+dynamics+of+multicellular+cell-cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.</a> Cell. 132, 487-498 (2008).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> Journal of visualized experiments : JoVE. , (2010).</li><li>Smalley, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulated+expression+of+zonula+occludens+protein-1+in+human+melanoma+associates+with+N-cadherin+and+contributes+to+invasion+and+adhesion.">Up-regulated expression of zonula occludens protein-1 in human melanoma associates with N-cadherin and contributes to invasion and adhesion.</a> The American journal of pathology. 166, 1541-1554 (2005).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+glutamine+transport+to+suppress+melanoma+cell+growth.">Targeting glutamine transport to suppress melanoma cell growth.</a> International journal of cancer. Journal international du cancer. 135, 1060-1071 (2014).</li><li>Lorenzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+division+dynamics+monitoring+in+3D+large+spheroid+tumor+models+using+light+sheet+microscopy.">Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy.</a> Cell division. 6, 22 (2011).</li><li>Pampaloni, F., Ansari, N., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+deep+imaging+of+live+cellular+spheroids+with+light-sheet-based+fluorescence+microscopy.">High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy.</a> Cell and tissue research. 352, 161-177 (2013).</li><li>Durand, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Hoechst+33342+for+cell+selection+from+multicell+systems.">Use of Hoechst 33342 for cell selection from multicell systems.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 30, 117-122 (1982).</li><li>Laurent, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroid+models+to+explore+cell+cycle+checkpoints+in+3D.">Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D.</a> BMC cancer. 13, 73 (2013).</li><li>LaRue, K. E., Khalil, M., Freyer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+proliferation+in+multicellular+spheroids+is+mediated+through+differential+expression+of+cyclin-dependent+kinase+inhibitors.">Microenvironmental regulation of proliferation in multicellular spheroids is mediated through differential expression of cyclin-dependent kinase inhibitors.</a> Cancer research. 64, 1621-1631 (2004).</li><li>Kyle, A. H., Huxham, L. A., Baker, J. H., Burston, H. E., Minchinton, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+distribution+of+bromodeoxyuridine-labeled+cells+is+strongly+dose+dependent.">Tumor distribution of bromodeoxyuridine-labeled cells is strongly dose dependent.</a> Cancer research. 63, 5707-5711 (2003).</li><li>Minchinton, A. I., Tannock, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+penetration+in+solid+tumours.">Drug penetration in solid tumours.</a> Nature reviews. Cancer. 6, 583-592 (2006).</li><li>Giesbrecht, J. L., Wilson, W. R., Hill, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiobiological+studies+of+cells+in+multicellular+spheroids+using+a+sequential+trypsinization+technique.">Radiobiological studies of cells in multicellular spheroids using a sequential trypsinization technique.</a> Radiation research. 86, 368-386 (1981).</li></ol>

PerkinElmer contributed to the costs of publication.

半自动化图像分析，鉴定球体内G1被捕区域，和增殖外层。这种方法可以在活球状体用光学部分被使用，或在固定的球体部分，以识别变化不仅在细胞周期但标记表达（通过免疫荧光），细胞死亡，或细胞形态在这些不同的区域。不同球状体区域内的细胞运动性，也可以定量 - 如果连同一个细胞跟踪图像分析​​步骤的现场共焦时间推移成像溶液。关键的图像分析是，高品质的Z-切片的共聚焦图像...

多细胞三维球状体已被被称为肿瘤模型数十年，但它只是在最近，他们已进入更常见的用法作为体外模型对许多实体癌症。它们越来越多地被用于高通量药物发现屏幕作为中间之间的复杂，昂贵和费时的体内模型和简单的，低成本的2D单层模型1-6。研究在2D培养往往无法在体内被复制。许多类型的癌症的球体模型是能够模仿的生长特性，药物敏感性，药物渗透，细胞-细胞相互作用，氧和营养物和坏死的发展是见于体内实体肿瘤6-11的有限的可及。球状体开发出坏死核心，包围芯静止或G 1被捕区域，并在球体7的周边增殖细胞。这些区域的发展可根据细胞密度，增殖率和球体12的尺寸而变化。据推测，见于这些不同子区的细胞异质性可能会导致癌症治疗电阻13,14。因此，分析细胞在这些区域中的能力分别是至关重要的理解肿瘤药物反应。 荧光泛素细胞周期指示符（FUCCI）系统是基于红色（Kusabira橙色-正）和绿色（Azami格林- AG）CDT1和geminin的荧光标记，这是降解的细胞周期15的不同阶段。因此细胞核出现红色于G1，黄S中的早期和绿色中的S / G2 / M期。我们在这里描述两个互补方法均使用FUCCI来识别细胞周期，随着使用成像软件或染料扩散流式细胞仪测定的，以确定细胞是否驻留在G1被捕中心或外细胞增生克环，和个体细胞从旋转椭圆体的边缘的距离。这些方法被开发在我们以前的出版物，在这里我们表明，黑色素瘤细胞在低氧区域中的旋转椭圆体的中心和/或在靶向治疗的存在下能够保持在G1期阻滞延长的时间段，并且可以重新在更有利的条件出现7进入细胞周期。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>agarose low melting point</td>
			<td>Life Technologies</td>
			<td>16520-050</td>
			<td>For sectioning</td>
		</tr>
		<tr>
			<td>noble agar&#160;</td>
			<td>Sigma</td>
			<td>A5431</td>
			<td>For making spheroids</td>
		</tr>
		<tr>
			<td>agarose for spheroids</td>
			<td>Fisher Scientific</td>
			<td>BP1356-100</td>
			<td>For making spheroids</td>
		</tr>
		<tr>
			<td>0.05% trypsin/EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin</td>
			<td>Sigma</td>
			<td>HT5014-1CS</td>
			<td>CAUTION: Harmful, corrosive. Use Personal Protective Equipment, do &#160;not breath fumes (open in a fume cupboard).</td>
		</tr>
		<tr>
			<td>live/dead near IR</td>
			<td>Life Technologies</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>vibratome</td>
			<td>Technical Products International, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>coulture cup</td>
			<td>Thermo-Fisher Scientific</td>
			<td>SIE936</td>
			<td>Mold for sectioning spheroids</td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Invitro</td>
			<td>FAL353072</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Sigma</td>
			<td>C5138&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer analyser</td>
			<td>Becton Dickinson</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>flowjo</td>
			<td>Tree Star</td>
			<td></td>
			<td>Flow cytometry software</td>
		</tr>
		<tr>
			<td>Vaccuum grease</td>
			<td>Sigma</td>
			<td>Z273554</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Vector Laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>FUCCI (commercial constructs)</td>
			<td>Life Technologies</td>
			<td>P36238</td>
			<td>Transient transfection only</td>
		</tr>
		<tr>
			<td>Cell strainer 70 um</td>
			<td>In Vitro</td>
			<td>FAL352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom 5 mL tubes (sterile)</td>
			<td>In Vitro</td>
			<td>FAL352003</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom 5 mL tubes (non-sterile)</td>
			<td>In Vitro</td>
			<td>FAL352008</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

有产生肿瘤球状体的几种方法，该协议使用非贴壁生长的方法，其中细胞在琼脂或琼脂糖3,7,9中培养。 图1示出 C8161黑素瘤球体的后琼脂上3天的例子。 C8161球状体形成正常尺寸的球体的直径为500 - 600微米（平均值= 565，标准差= 19中，n = 3）后3天。其他黑素瘤细胞系，将形成球状体包括：WM793，WM983C，WM983B，WM164，1205LU（用该细胞系形成的球状体是不规则的，较不密集19）。...

Watch this Scientific Journal Video about Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids at JoVE.com

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

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.

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

imaging-flow-cytometry-based-analysis-cell-position-cell-cycle-3d

Research

JoVE Journal

Medicine

12.9K 次观看.  The Centenary Institute. 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. 

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

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 ...

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 ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;：单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

骨关节炎模型中骨髓、骨髓、淋巴结和合成组织内免疫细胞子集的流动细胞测量分析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model

Apolipoprotein E (Apoe)- or low density lipoprotein receptor (Ldlr)-deficient hyperlipidemic mice are the two most commonly used models for atherosclerosis research. They are used to study the impact of a various genetic factors and different cell types on atherosclerotic lesion formation and as well as test the development of new therapies. Isolation, excision of the whole aorta, and quantification of Oil Red O-stained atherosclerotic lesions are basic morphometric methods used to evaluate atherosclerotic burden. The goal of this protocol is to describe an optimized, step-by-step surgical method to dissect, perfuse-fix, isolate, stain, image and analyze atherosclerotic lesions in mouse aortas with Oil Red O. Because atherosclerotic lesions can form anywhere in the entire aortic tree, this whole aorta Oil Red O staining method has the advantage of evaluating lipid-laden plaques in the entire aorta and all branches in a single mouse. In addition to Oil Red O staining, fresh isolated whole aortas can be used for variety of in vitro and in vivo experiments and cell isolations.

This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.