这项工作是由芝加哥妇女委员会的大学，温迪威尔案例基金和芝加哥综合癌症中心支援津贴大学（P30 CA014599）的支持。我们要感谢在芝加哥大学的流式细胞仪核心提供的帮助在建立这个实验，并且还有德容实验室的其他成员，特别是莱斯利·佩德拉萨和肖恩·麦康诺。

与斑马鱼的所有程序已批准的机构动物护理和使用委员会（IACUC）在芝加哥大学。芝加哥大学是由协会为实验动物评估和认可委员会（AAALAC）的认可。 1.生成并分离荧光标记的T-ALL中斑马鱼细胞<ol><li> 显微注射进同源斑马鱼胚胎 <ol><li>使用市售试剂盒，隔离圆形RAG2:c-myc和RAG2:绿色荧光蛋白（RAG2:GFP）含有末端反向重复的质粒DNA通过TOL2转座（参见材料的表用于质粒DNA的源）12识别并洗脱DNA在无菌水中。 注:GFP在这项研究中使用，但任何荧光蛋白会工作。 </li><li>制备PCS2-转座（TOL2）RNA <ol><li>使用市售试剂盒隔离TOL2质粒DNA（见吨材料的质粒DNA的来源的他表）12和洗脱在无菌水中的DNA。 </li><li>孵育TOL2 DNA的1微克，在37℃用在适当的缓冲液以线性化质粒5个单位的NotI限制性内切酶的1个小时。热灭活的NotI在65℃下20分钟。 </li><li>转录从使用市售的RNA转录试剂盒13中的线性化DNA SP6 RNA聚合酶的TOL2 RNA。在-80℃下储存在单次使用的等分试样的RNA。 </li></ol></li><li> c-myc基因的质粒DNA，RAG2的250ng的:GFP的质粒DNA，RNA TOL2 150纳克，和酚红的0.5μL加入250ng的RAG2的制备在冰上注射组合。将音量调整到10微升无核酸酶的水。 </li><li>注入注射混合物的〜1纳升成单细胞阶段的同基因斑马鱼胚胎的细胞使用微量注射器和针头从玻璃毛细管拉动时，按照先前在ESTAB发现方法8,14。 注意:通过孤雌生殖15产生的同基因斑马鱼在这里用于消除移植后的遗传异质性和免疫排斥反应。然而，也可以使用野生型斑马鱼或任何所需的菌株。 </li><li>在注射后24小时和48小时内清除死亡，不发达或畸形的胚胎16 。 </li><li>注射后6天将注射的鱼放在鱼系统上，并按照标准方案6提高。 </li></ol></li><li> 筛选注射斑马鱼并监测肿瘤生长 <ol><li>注射后21天，使用荧光显微镜（激发带通:470/40，发射带通:525/50） 8将一只注射的幼虫置于培养皿中的胚液培养基液滴中并筛选GFP荧光。 注意:斑马鱼幼虫hAVE集成两个RAG2:c-myc和RAG2:GFP将具有绿色荧光胸腺（阳性鱼），并且可以显示出已经超出了胸腺恶性肿瘤绿色荧光细胞的扩增。用GFP +胸腺所有的鱼会继续发展成一个GFP + T-ALL。不证明质粒的整合幼虫不会有任何绿色荧光的细胞（负鱼）。 </li><li>分离负鱼（基于缺少荧光的）到不同的罐，并在28天和35天注射后再次监视鱼。在28天和35天注射后，麻醉鱼用0.02％（MS-222）在鱼系统水之前筛选缓冲三卡因。 </li><li>将每个斑马鱼幼虫结果为阳性的荧光标记的胸腺或用于监测肿瘤的生长，每周三次的个体罐荧光标记的肿瘤。 </li></ol></li><li> 收集的荧光标记的T-ALL细胞从原发肿瘤鱼 <ol><li>制备至少20 0.9倍磷酸盐缓冲盐水（FBS / PBS）10％热灭活的胎牛血清中的溶液。 </li><li>加入1毫升FBS / PBS中，以盐肝素钠的一个管形瓶（300个单位）。 </li><li>选择鱼轴承在身体的超过50％的肿瘤，或者具有进食困难或游泳那些鱼。用三卡因或冰水浸泡过量他们牺牲的鱼。确认通过鳃运动和心跳的缺乏死亡。 </li><li>用刀片除去鱼的头部。 </li><li>洗鱼的空腔与肝素溶液以除去过量的红血细胞。 <ol><li>使用移液管，注入100微升肝素溶液（在步骤1.3.2的方法制备）成鱼的体腔内。去除溢出的所有液体（它将包含血细胞），并将其移液到1.5mL微量离心管中。 </li><li>选用一个新鲜枪头再次冲洗身体。执行至少三次洗涤，或直到溢出从液体出现免费BL的洪水。 </li><li>从在步骤1.3.5.1和1.3.5.2洗液丢弃所有液体，因为它不会在进一步的分析中使用。 </li></ol></li><li>收集白血病细胞。 <ol><li>注入100μL的FBS / PBS的成鱼的体腔内。 </li><li>轻轻推压到鱼与移液管尖端的主体的外侧以挤压/压出的肿瘤细胞。 注意:如果使用解剖显微镜执行该步骤，所述肿瘤细胞溢出体腔将是显而易见的。 </li><li>收集液体和细胞从每个洗到1.5mL微量离心管中。 </li><li>重复步骤1.3.6.1-1.3.6.3直到大部分肿瘤细胞被收集（通常5-10倍）。收集所有的肿瘤细胞在同一个1.5毫升微量离心管中。使细胞保持在室温下。 </li></ol></li><li>轻轻吹打它们以解离细胞的团块混合来自步骤1.3.6收集到的肿瘤细胞。通过4过滤细胞悬浮液0-μm筛网。用100μLFBS / PBS清洗过滤器1-2次。将细胞保持在室温。 </li><li>将2μL细胞悬浮液与18μL台盼蓝（0.4％，1:10稀释，并过滤）在新管中混合。在血细胞计数器上加载10μL，计数活（非蓝）荧光细胞数，计算分离的肿瘤细胞数。 注意:非蓝色GFP阴性细胞可能是正常的，非恶性的T细胞，不应该计数。使用Hoechst 33342染色细胞需要至少2×10 6个细胞。 </li></ol></li></ol> 2.用Hoechst 33342染色荧光标记的T-ALL细胞以鉴定侧面群体<ol><li>稀释的Hoechst 33342 1:10用无核酸酶的H 2 O制成1mg / mL的工作浓度。将染料在4℃和黑暗中储存。 </li><li>通过将49.1mg溶解在1mL二甲基亚砜（DMSO）中来制备维拉帕米的100mM溶液。 注:维拉帕米是ABC转运的抑制剂，并且是侧群实验的重要控制。添加维拉帕米样品会抑制ABC转运至外排赫斯特33342染料的能力。因此，当维拉帕米加入到样品真实的一面人口将消失。 </li><li>设定水浴至28℃。 </li><li>通过添加细胞和解决方案，荧光激活细胞分选仪（FACS）管制备在黑暗中的样品和对照。 <ol><li>通过加入10个6个细胞，/ 15μL的1毫克姆·霍赫斯特33342，和2.5微升DMSO的至FACS管中制备样品。调整音量，以1毫升FBS / PBS。 </li><li>通过加入10个6个细胞，/ 15μL的1毫克姆·霍赫斯特33342，和100mM的2.5μL维拉帕米至FACS管中制备的对照。调整音量，以1毫升FBS / PBS。 注:至少10个6个细胞的/推荐的样品，但染色更多的细胞是优选的，特别是如果侧populat离子电池将被排序以用于进一步分析。不超过10个6个细胞/毫升，并保持在15微克/毫升的Hoechst的33342浓度。如果10×10 6个细胞染色时，总反应体积为10毫升。 </li></ol></li><li>在孵育在黑暗中120分钟的28℃水浴中。 </li><li>培养结束后，立即置于冰上的细胞，并让他们在黑暗中。 </li><li>沉淀细胞在4℃下在300×g下6分钟。除去上清液。用1mL FBS / PBS清洗。 </li><li>沉淀细胞在4℃下在300×g下6分钟。除去上清液。重悬细胞在1mL FBS / PBS中。 </li><li>保持在4℃直至细胞分选。 </li><li>到的细胞分选，加入2μL的碘化丙啶（PI）的库存（1毫克/毫升），以每1毫升FBS / PBS的15分钟之前（终浓度:2微克/毫升）。拌匀。 </li></ol> 3.使用FACS找到荧光标记的T-ALL细胞群中侧群<ol><li&gt;在配备有UV激光的Hoechst 33342染料的激发和488纳米激光器为PI激发和用于检测GFP + T-ALL细胞的细胞分选仪分析染色的斑马鱼T-ALL细胞悬浮液。 </li><li>确定门的活，GFP + T-ALL细胞。 <ol><li>使用前向散射（FSC）和侧向散射（SSC）区与栅极出碎屑。 注:FSC是用来区分细胞大小和SSC用于区分细胞的粒度。 </li><li>使用FSC和SSC宽度歧视双峰。 注意:有通过流式细胞仪检测器确定了三个部分组成:面积，高度，和宽度。区域通常用于测量细胞的荧光。宽度测量的飞行时间，两倍宽，如果两个小区时聚集在一起。 </li><li>对于活肿瘤细胞，绘制使用适当的检测器周围的正面为GFP和阴性PI细胞的栅极（ 例如 ，FITC对GFP和PerCP Cy5-5为PI）。 </li></ol></li><li&gt;在活细胞门中，通过Hoechst Blue和Hoechst Red带通滤光片（分别为450/50和670/30）测量Hoechst 33342荧光。 注意:如果存在，侧面人群在建立之后将是明显的。重要的是，侧面人群必须在维拉帕米控制中消失，才能将其视为真正的人口。 </li><li>如果对侧面细胞进行分选以进一步实验，则使用大的喷嘴尖端（100μm），并将细胞收集到含有3 mL 100％FBS的FACS管中。使用较大的喷嘴尖端（100μm）而不是较小的尖端（70μm），以提高分选后的细胞活力。 </li></ol>

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</ol>

作者没有什么可以披露的。

侧群测定法是高度敏感的;因此，该协议的微小变化可导致在检测侧群细胞是困难的。首先，在染色步骤中的温度是特定于每个动物/细胞系统。对于哺乳动物系统中，侧群测定典型地在37℃下2进行。当斑马鱼T-ALL细胞在37℃孵育，许多细胞死亡，这使得在该温育温度不可接受（数据未示出）。当小林和他的同事（2008），斑马鱼解剖肾髓的侧群分析中，温育在25℃，5进行。在该温育温度不足以用于斑马鱼T-ALL细胞，因为它导致了弱的Hoechst 33342轮廓（数据未示出）。在这里描述的协议，28℃的温育温度是最佳的，以保持细胞生存力和实现强劲的Hoechst 33342轮廓。 为侧群测定，需要考虑与现有的测试第二个条件是Hoechst公司33342的浓度如果赫斯特33342的浓度过低时，侧群可能不可见，或侧群的表型（染料流出能力）可被错误地增加，识别多个侧群细胞比实际存在2。在另一方面，过高的Hoechst 33342的浓度可能是有毒的细胞。斑马鱼T-ALL细胞，15微克/姆·霍赫斯特33342孵育120分钟是最佳的组合。赫斯特33342的低浓度被尝试用于斑马鱼T-ALL细胞，但侧群并不总是明显的（数据未示出）。的90分钟的总孵育时间为足以检测侧群，但之后的温育时间120分钟观察侧群细胞的最大数目。 正如前面哺乳动物员的报告<html> ystems，并不是每个肿瘤都会有可检测的边群17,18,19 。使用这种方法，我们观察到17个T-ALL测试中的14个（数据未显示）。从T-ALLs分离的侧面群体细胞，这种方法预期适用于其他斑马鱼肿瘤模型。 在异质斑马鱼T-ALL肿瘤内，白血病起始细胞（LIC）能够重新启动肿瘤生长，因此被认为是导致癌症复发和转移的原因。目前，斑马鱼没有方法可以在体内预期分离LIC。计算LIC频率需要有限稀释的肿瘤细胞移植8,10。以前发表的克隆斑马鱼研究已经发现，LIC占原代T-ALL细胞的0.1-1.4％"外部参照"&gt; 10。在此，在从克隆斑马鱼T-承滴盘侧群细胞（0.2-1.0％）的频率类似的变异被报告（ 图2）。进一步的实验，需要确定在低收入国家侧群是否富集;然而，LIC频率的范围和侧群细胞在这些T承滴盘的百分比之间的相似性支持这一假设。 而侧人口分析是敏感的，并不总是很容易进行，该测定法允许研究人员，对于第一次，在斑马鱼细胞的罕见群体分离T-ALL可具有干细胞或祖细胞的性质。目前没有在斑马鱼中，以确定茎或祖细胞样细胞可用细胞表面标记物;因此，分离出侧群的能力是非常有前途的。该方法的未来应用包括排序的侧群T-ALL细胞的有限稀释移植实验，以评估L的富集集成电路。此外，这种方法将允许限定低收入国家特性待阐明，包括支配在斑马鱼T-ALL低收入国家的分子机制。这些实验可以为新的治疗靶点的发现为T-ALL提供了出发点。

侧群体测定利用细胞膜上的ATP结合盒（ABC）转运蛋白的高水平来增强组织内某些细胞的能力，从而排出DNA结合染料Hoechst 33342。使用UV激光激发染料后，可以使用双波长流式细胞术分析鉴定外排Hoechst 33342染料的细胞。该测定首先用于鉴定鼠造血干细胞（HSCs） 1 ，但是它已被用于鉴定许多组织和癌症中的干细胞/祖细胞群（参见参考文献2）。然而，并不是所有的细胞群均具有侧面群体，并且并不是所有的侧面群体都富含茎/祖细胞。 斑马鱼是一种强大的脊椎动物遗传模型系统，用于研究人类癌症3,4 ，具有优于传统小鼠的许多优点癌症的皮肤。斑马鱼胚胎在外部受精并且是光学透明的，促进转基因和体内观察病理过程，包括癌症起始和进展。迄今为止，用于检测潜在的干细胞或祖细胞的侧面群体测定仅已经应用于斑马鱼中的肾脏骨髓来识别HSC，而不是任何斑马鱼癌模型5,6 。 T细胞急性淋巴细胞白血病（T-ALL）的斑马鱼模型与人类T-ALL 7,8,11的形态学和遗传学相似。 T-ALL是一种侵略性恶性肿瘤，在人类中占儿童的10-15％，成人ALL的占25％。虽然T-ALL的治疗有所改善，但复发仍然很常见，预后不良。 T-ALL肿瘤是异质的并含有许多不同的肿瘤细胞亚群，包括白血病起始细胞（LIC）。 LIC通过其从单个细胞再生整个肿瘤的能力来定义，并且可以通过有限稀释移植测定（LDA）将不同的细胞剂量移植到受体中来计算肿瘤细胞群体内的LIC的频率。虽然已经在斑马鱼中进行LDA实验以计算LIC 8,10,11的频率，但是这种确定是事后做出的，并且不允许LIC的预期分离。因此，缺乏前瞻性分离富集癌症干细胞活性的群体的方法。识别和分离侧群体细胞与斑马鱼T-ALLs是解决这一缺陷的第一步。 这里提出的方案描述了如何有效地产生斑马鱼的T-ALL肿瘤afish利用TOL2介导的转基因，收获T-ALL肿瘤细胞，并用Hoechst 33342染色他们能够识别细胞的侧群。未来与斑马鱼T-ALL 体内实验可以包括侧群细胞是否被富集低收入国家或有其他茎或祖样性质。

<table><tbody><tr><td>Syngeneic zebrafish (CG2)</td><td></td><td></td><td>See Mizgireuv &lt;em&gt;et al.&lt;/em&gt;, 2006</td></tr><tr><td>&lt;em&gt;rag2&lt;/em&gt;:c-myc plasmid</td><td></td><td></td><td>Langenau Laboratory</td></tr><tr><td>&lt;em&gt;rag2&lt;/em&gt;:GFP plasmid constructed in de Jong Lab</td><td></td><td></td><td>&lt;em&gt;rag2&lt;/em&gt; promoter from Langenau Laboratory</td></tr><tr><td>pCS2-transposase plasmid</td><td></td><td></td><td>Chien Laboratory</td></tr><tr><td>NotI -HF restriction enzyme</td><td>New England Bio-Labs Inc.</td><td>R3189S</td><td>500 units in 25 &amp;micro;L</td></tr><tr><td>mMessage Machine SP6 Transcription Kit</td><td>Ambion</td><td>Thermo Fisher Scientific - AM1340</td><td>25 reactions</td></tr><tr><td>QIAGEN Plasmid Midi Kit</td><td>Qiagen, Inc.</td><td>12643</td><td></td></tr><tr><td>SteREO Discovery V8 Microscope</td><td>Carl Zeiss Microscopy</td><td>495015-0008-000</td><td></td></tr><tr><td>Digital microinjector</td><td>Tritech Research</td><td>MINJ-D</td><td></td></tr><tr><td>Brass straight-arm needle holder</td><td>Tritech Research</td><td>MINJ-4</td><td></td></tr><tr><td>Magnetic base and stand</td><td>Tritech Research</td><td>MINJ-HBMB</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige International</td><td>MN153</td><td></td></tr><tr><td>Glass capillaries</td><td>Sutter Instrument Co.</td><td>B100-50-10</td><td>10 cm without filament</td></tr><tr><td>Flaming/Brown micropipette puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf North America Inc.</td><td>930001007</td><td></td></tr><tr><td>Phenol Red solution</td><td>Sigma Life Science</td><td>P0290</td><td>0.5% concentration 100 mL</td></tr><tr><td>Plastic Transfer pipettes</td><td>Fisherbrand</td><td>137119D</td><td>graduated 7.5 mL bulb</td></tr><tr><td>Tricaine methanesulfonate (MS-222)</td><td>Western Chemical, Inc.</td><td>Fisher Scientific - NC0342409</td><td>100 g</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>HyClone Laboratories, Inc.</td><td>Fisher Scientific - SH3007103</td><td>500 mL</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Gibco by Life Technologies</td><td>1001-023</td><td>pH 7.4 (1x) - 500 mL</td></tr><tr><td>Heparin sodium salt</td><td>Sigma-Aldrich</td><td>2106</td><td>15 - 300 unit vials</td></tr><tr><td>40 &amp;micro;m mesh filter</td><td>Falcon Corning Brand</td><td>352340</td><td>Case of 50</td></tr><tr><td>Trypan blue</td><td>Sigma Life Science</td><td>T8154</td><td>20 mL - Solution (0.4%)</td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td>10 mL</td></tr><tr><td>Verapamil</td><td>Sigma Life Science</td><td>V4629</td><td>1 g</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma Life Science</td><td>D8418</td><td>100 mL</td></tr><tr><td>Propidium Iodide</td><td>Life Technologies</td><td>P3566</td><td>10 mL</td></tr><tr><td>FACSAria Fusion cell sorter</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva 8.0.1</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo v10.2</td><td>FlowJo, LLC</td><td></td><td></td></tr></tbody></table>

isolation of the side population in myc-induced t-cell acute lymphoblastic leukemia in zebrafish

异质细胞群，从任一健康或恶性组织，可以含有其特征在于，外排至DNA结合染料赫斯特33342细胞的这种"侧群"的差分能力的细胞群可使用的Hoechst 33342后流式细胞方法来鉴定染料由紫外线（UV）激光激发。许多细胞类型的侧群包含茎或祖细胞样细胞。然而，并非所有类型的细胞具有可识别的侧群。 斑马鱼 ，斑马鱼， 在 T细胞急性淋巴细胞性白血病（T-ALL）的体内模型中一个健壮的，但这些斑马鱼T-承滴盘是否具有侧群是未知的。这里所描述的方法概述了如何分离在斑马鱼T-ALL侧群细胞。首先，经由TOL2质粒的显微注射到单细胞阶段的胚胎产生的T-ALL在斑马鱼。一旦肿瘤已发展到一个阶段，在其业务拓展到超过一半的ANI的马氏体，T细胞可以收获。然后将细胞用Hoechst 33342染色，并通过流式细胞术检查侧群细胞。该方法在斑马鱼T-ALL研究中有广泛应用。虽然斑马鱼中没有已知的细胞表面标志物证实这些侧面细胞是否是癌症干细胞样的，但是体内功能性移植测定是可能的。此外，单细胞转录组学可以应用于鉴定这些侧群体细胞的遗传特征。

为了有效地产生荧光myc基因诱导的T-ALL在斑马鱼中的肿瘤，通过转座TOL2位点侧接的环状DNA构建体可以被共注射TOL2 RNA。从注射线性化DNA引入斑马鱼胚胎以前的研究报告，5％的转基因率8。与协议调整包括TOL2介导的转基因，转基因率从10-44％，可观察到（ 表1）。 在代表性例子中，与GFP + T-ALL鱼用Hoechst 33342染色以确定细胞在侧群的百分比。 图1示出用于确定侧群细胞的百分比的选通方案。首先，使用FSC和SSC中，T-ALL细胞通过排除碎片（ 图1A），并随后选择对双重峰分离（ 图1B）。接下来，通过对GFP +进行门控细胞分离所有活细胞，并通过死细胞鉴别器PI（ 图1C ）分离阴性细胞。一旦选择了单个活的T-ALL细胞，侧面群体可以在Hoechst 33342轮廓中找到。在主细胞群中，Hoechst 33342保留在细胞中，这些细胞可以通过DNA含量分成组。然而，由于细胞膜上的ABC转运蛋白数量较多，侧面细胞外排染料，细胞的侧面群体与主要细胞群体区分开。为了可视化侧面人口，Hoechst轮廓用于比较两个通道，Hoechst Blue和Hoechst Red。侧群体是从主细胞群体的左侧朝向Hoechst蓝色轴线延伸的细胞的暗淡的尾巴（ 图1D ）。 当细胞用抑制剂治疗时，真正的人群将会丢失˚FABC转运，如维拉帕米。 图2示出几个代表性T-ALL肿瘤侧种群，以及当这些肿瘤用Hoechst 33342染色过程中与维拉帕米处理以确保样品中发现的人口是一个真正的侧群如，那些细胞必须消失在抑制剂的存在下，如示于图2。 <table height="525" width="487"><tbody><tr><td> 库存数量 </td><td> 的鱼屏蔽 </td><td> 一些积极 </td><td> 转基因率 </td></tr><tr><td> 930 </td><td> 21 </td><td> 3 </td><td> 14.3％ </td></tr><tr><td> 934 </td><td> 12 </td><td> 4 </td><td> 33.3％ </td></tr><tr><td> 950 </td><td> 12 </td><td>五</td><td> 41.7％ </td></tr><tr><td> 951 </td><td> 19 </td><td> 3 </td><td> 15.8％ </td></tr><tr><td> 960 </td><td> 3 &lt;/ TD&gt; <td> 1 </td><td> 33.3％ </td></tr><tr><td> 983 </td><td> 9 </td><td> 4 </td><td> 44.4％ </td></tr><tr><td> 1004 </td><td>三十</td><td> 3 </td><td> 10.0％ </td></tr><tr><td> 1013 </td><td> 4 </td><td> 1 </td><td> 25.0％ </td></tr><tr><td> 1025 </td><td> 35 </td><td>五</td><td> 14.3％ </td></tr><tr><td> 1029 </td><td> 10 </td><td> 2 </td><td> 20.0％ </td></tr><tr><td> 1065 </td><td>五</td><td> 1 </td><td> 20.0％ </td></tr><tr><td> 1070 </td><td> 6 </td><td> 1 </td><td> 16.7％ </td></tr><tr><td> 1073 </td><td> 19 </td><td> 8 </td><td> 42.1％ </td></tr><tr><td> 1079 </td><td> 8 </td><td> 1 </td><td> 12.5％ </td></tr><tr><td> 1105 </td><td> 38 </td><td> 7 </td><td> 18.4％ </td></tr><tr><td> 1193 </td><td>三十</td><td> 8 </td><td> 26.7％ </td></tr><tr><td> 1215 </td><td> 9 </td><td> 1 </td><td> 11.1％ </td></tr><tr><td>1229 </td><td> 10 </td><td> 2 </td><td> 20.0％ </td></tr><tr><td> 1314 </td><td> 6 </td><td> 1 </td><td> 16.7％ </td></tr></tbody></table> 表1:使用tol2转座子质粒显微注射到斑马鱼胚胎时的转基因率。计算出的转基因速率为十九个单独的注射天数。筛选的鱼数表示注射后21天仍然存活的鱼数，数量阳性代表荧光肿瘤阳性的鱼数。 <img alt="图1" src="/files/ftp_upload/55711/55711fig1.jpg" /> 图1:侧面人口门控方案。用于鉴定斑马鱼中GFP + T-ALL中的侧群的门控方案的一个例子。 （ A ）首先，使用前向散射（FSC）和侧向散射（SSC）的区域。 （B）接着，单个细胞通过歧视使用FSC和SSC宽度参数双重隔离。 （C）由于本例是肿瘤GFP +，所有活细胞必须是死细胞标记物碘化丙啶GFP +和负。 （D）的侧群可以与Hoechst的红与蓝的Hoechst轮廓中看到。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55711/55711fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55711/55711fig2.jpg" /> 图2:侧群消失当与ABC转运蛋白抑制剂，维拉帕米治疗。为侧群盖茨所示。顶部面板显示四个不同的肿瘤样本。需要注意的是并不是所有的肿瘤具有相同的侧群比例和侧群CA是很重要的Ñ寻找测试的每个肿瘤不同。底部面板显示，用Hoechst 33342染色以阻止染料流出期间与维拉帕米治疗的肿瘤样品。而侧种群可能看起来不同对于每个肿瘤，维拉帕米的效果是一样的:侧群消失。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55711/55711fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish at JoVE.com

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

在这里，我们描述了一种从myc诱导的T细胞急性淋巴细胞白血病（T-ALL）的斑马鱼模型中分离侧群体细胞的技术。这种侧群体测定是高度敏感的，描述为斑马鱼T-ALL，但它可能适用于其他恶性和非恶性斑马鱼细胞类型。

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

isolation-side-population-myc-induced-t-cell-acute-lymphoblastic

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

6.6K 次观看.  The University of Chicago. 在这里，我们描述了一种从myc诱导的T细胞急性淋巴细胞白血病（T-ALL）的斑马鱼模型中分离侧群体细胞的技术。这种侧群体测定是高度敏感的，描述为斑马鱼T-ALL，但它可能适用于其他恶性和非恶性斑马鱼细胞类型。 

视频: Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

发育生物学

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

<meta charset="utf8"></head><body>使用细胞增殖染料分选估计斑马鱼 T-ALL 中的 LSC 频率

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry.
Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments.
Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

<meta charset="utf8"></head><body>作者聚焦:从斑马鱼 T-ALL 中鉴定和分离静止白血病干细胞

We used cell proliferation staining to identify quiescent cells in the zebrafish T-acute lymphoblastic leukemia model. The stain is retained in non-dividing cells and reduced during cell proliferation, enabling the selection of dormant cells for further interrogation. This protocol provides a functional tool to study self-renewal in the context of cellular quiescence.

使用细胞增殖染色鉴定斑马鱼 T 细胞急性淋巴细胞白血病模型中的静止细胞 

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect ...

癌症研究

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

生物学

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish

Transgenic lck:eGFP zebrafish express GFP highly in T lymphocytes, and have been used to study T cell development and acute lymphoblastic leukemia. This line can be used to study B cells, which express lck at lower levels. This protocol describes purification of malignant and non-malignant B cells from lck:eGFP zebrafish.

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and ...

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼

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V3的HIV - 1取向的基因型推理使用人口为基础的测序

分离和斑马鱼胚胎单细胞的表征

移植物抗宿主病的诱导和

T细胞实时成像实时信号复合物的形成

使用细胞增殖染色鉴定斑马鱼 T 细胞急性淋巴细胞白血病模型中的静止细胞

使用细胞增殖染色鉴定斑马鱼 T 细胞急性淋巴细胞白血病模型中的静止细胞

使用细胞增殖染色鉴定斑马鱼 T 细胞急性淋巴细胞白血病模型中的静止细胞

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

从 lck 中分离恶性和非恶性 b 细胞: egfp斑马鱼