Name: isolation and characterization of single cells from zebrafish embryos
Uploaded: 2016-03-12T15:00:00.000+00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

We thank Dr. C. Geoffrey Burns for fish stock. The authors are grateful to UNC Flow Cytometry Core Facility, UNC-CGIBD AAC core for resources enabling this project, and the ZAC facility for animal care. L.S. is supported by NIH T32 grant HL069768-13 (PI, Nobuyo Maeda). N.F. is supported by NSF Graduate Research Fellowship NSF-DGE-1144081. This study was supported by NIH P30DK034987 grant (to UNC Advanced Analytics Core), American Heart Association Scientist Development Grant 13SDG17060010 and Ellison Medical Foundation New Scholar Grant AG-NS-1064-13 (to Dr. Qian), and NIH R00 HL109079 grant (to Dr. Liu).

该协议要求使用的活，成年斑马鱼产生的胚胎。将胚收获组织收集。重要的是要获得相应的伦理审查委员会批准进行这项实验。 1.获取胚胎上演<ol><li>在实验前一天，繁殖准备健康，成年斑马鱼。放置一个男性和一个养殖池一个明确的分隔两侧一名女性。 </li><li>重复1.1所必需的足够胚胎生产下游应用尽可能多的养殖池。获得从野生型鱼和转基因鱼胚胎表达的感兴趣的细胞类型的荧光蛋白质。 注意:需要在步骤2-8下游应用胚胎数依赖于在感兴趣的时间点感兴趣的细胞的相对丰度。虽然这可能通过细胞类型而异，200胚胎产生2,000-5,000排序细胞当C感兴趣的厄尔代表&lt;细胞总数的1.0％，在24 HPF（小时受精后）。 </li><li>第二天早上，通过转移鱼到一个新的养殖池在面包屑槽换水和删除的分隔符。倾斜槽以一定的角度，鼓励养殖。 </li><li>收集上演胚胎。 <ol><li>每15分钟，由成年人转移到一个新的养殖池，并通过这些留守通过滤茶鸡蛋收集胚胎。 注意:当保持在可比的密度和温度的斑马鱼胚胎的同步发展。 </li><li>冲洗（在1L双蒸水0.21克/ L的即时海洋盐）与蛋水鸡蛋和转移到陪替氏培养皿。与空气循环转移培养皿在潮湿培养箱中于28.5℃。 </li></ol></li><li>最后收集后两小时，排序受精，多细胞胚胎成10cm陪替皿并降低密度，以每皿50胚胎。从一个单一的，15选择胚胎用于下游应用集合分钟时间窗口。孵育28.5℃的胚胎。 注:例如，在8点半，8时45分，9:00 9:15，9:30，9时45，10:00，上午10点15分收集胚胎。跨越时间点相比较，如果受精胚胎人数最多的是从上午9时收集到的离合器，然后只使用这些胚胎下游应用。 </li></ol> 2.设置单细胞分离<ol><li>感兴趣（18 HPF）的时间点前约30分钟，从他们的绒毛膜手动用细镊子取出胚胎。 </li><li>收集并标记为每个条件如下:2发2 ml微量离心管一40微米的细胞过滤网，一次35 MM细胞培养皿中，并用一个35微米的细胞滤网淋上2 FACS管。 </li><li>冰浴的下列试剂:含有蛋水0.21克/ L即时海洋盐1L双蒸水;脱yolking含有55 mM氯化钠，1.8 mM的KCl和1.25毫碳酸氢钠缓冲液;和FACS缓冲液含有Leibovitz的L-15补充了5％热灭活的胎牛血清的介质。 </li><li>每带细胞分离试剂1的样品1毫升RT。解冻1毫升细胞分离试剂2每个样品上冰。把使用前立即RT。 </li></ol> 3.单细胞分离<ol><li>使用大口径玻璃吸管，转移100-300胚胎2ml的微量离心管中蛋水的最小量。 </li><li>通过在冰上用1ml冰冷的蛋水更换水和浸没管20分钟安乐死胚胎。 注意！关键是要取得适当的体制动物保护监督委员会批准，该安乐死方法（在冰上冷却后细胞分离二次安乐死）。标准安乐死通常需要胚胎&lt;3天受精后它们被冷却后，这是不恰当的用于获得活细胞漂白。 </li><li>用1毫升的冰冷蛋水冲洗胚胎两次。要洗，使用大口径玻璃吸管取出鸡蛋水和P1000加入鸡蛋水。 </li><li>通过用1ml Deyolking缓冲器，带有一个P1000尖更换胚胎水和捣碎8-12次去除蛋黄，或直到蛋黄溶解并只胚胎的机构是可见的。 </li><li>收集通过离心组织在300×g离心1分钟。用吸管轻轻地去除上清液而不破坏组织的沉淀。在1毫升鸡蛋水再挂起。 </li><li>重复步骤3.5，共洗涤三次，但在最后的洗涤，在1毫升的RT细胞解离试剂1重新暂停。 </li><li>孵育在细胞解离试剂1在RT 10分钟与管水平放置。每2-3分钟，轻轻地用吸管P1000防止结块磨碎。 注:在研磨步骤，轻拿轻放样品。有时一个单一的，大丛将形成胚体纠结管。这会与温和的研磨辅助附加消化分散。不要旋涡振荡。 </li><li>离心300 XG 3分钟收集组织。在1毫升细胞分离试剂2取出上清，重悬。 </li><li>在室温下孵育5-15分钟。水平放置的管子。每2-3分钟，轻轻磨碎，以防止结块。每5分钟，评估消化的进展。 <ol><li>为了评估消化进度，稀释2微升上清到18微升FACS缓冲液和吸管作为液滴到细胞培养皿。 </li><li>将盖玻片在样品和10X 20X和放大倍率组织培养显微镜下观察。 注:编制应显示为单细胞，小集群，大集群，和偶尔的大多是原封胚胎体的混合。 </li><li>视觉评估是单个细胞和小簇的制剂的比例。 注:在消化会减少可行性;在消化会减少单细胞产量。&lt;/ LI&gt; </ol></li><li>收集通过离心细胞制剂在300×g离心5分钟。弃去上清液并在1ml再悬浮冷FACS缓冲液。 </li><li>用浸湿FACS缓冲液40微米的细胞过滤。穿过40微米的细胞过滤细胞悬液到35毫米的细胞培养皿。用1ml的FACS分选缓冲液洗细胞过滤一次。 </li><li>转移流通到2ml微量离心管中。在300 xg离心收集细胞离心5分钟，并在100μlFACS缓冲液重悬浮。 </li><li>算使用血球细胞产量。进一步稀释样品建议选择的FACS机器每毫升，或最佳浓度5×10 6个细胞。可选当在血球计数细胞产量，用锥虫蓝染剂死细胞以确认之前FACS分选的细胞制剂的可行性。 注:准备被过度稀释，将需要大量的时间过量的排序。准备工作过于Çoncentrated易于聚集成多聚体，并为次优排序纯粹的人口。 </li><li>储备各试样的10〜20％，以使用作为未染色对照。对于剩余的样品，加入染色死细胞荧光活/死（L / D）的歧视染料到每个样品。不洗。 注:穿透细胞用的核酸可以被用作的L / D染料受损细胞膜和污渍不限荧光染料，只要该荧光光谱是与感兴趣的选择和荧光标记的细胞的流式细胞仪机兼容。 <ol><li>加入染料的一些细胞会在运输途中死FACS净化，应从排序人口被排除后，不要洗的准备。 </li></ol></li><li>皑皑浸湿FACS管20微升FACS缓冲的35微米的细胞过滤。加入细胞的过滤器，并通过重力收集。存储在冰上。 </li></ol> 4. FACS富集<ol><li>使用FACS来富集单，表达荧光标记的活细胞。 <ol><li>设置一个门上的前向散射（FSC-A）幅度VS侧散射幅度（SSC-A）的散点图从碎片区分细胞，都与线性缩放。 注意！斑马鱼细胞通常比小鼠或人类细胞小。这反映在细胞和碎片之间的下基底分离。 </li><li>从在4.1.1设置栅极，设置一个门以富集单细胞和排除使用前向散射高度（FSC-H）和低侧散射高度（SSC-H）的散点图多体，既具有线性缩放。 注:具有不成比例的高FSC-H和低SSC-H细胞可能多聚体和排序被排除在外。 </li><li>从4.1.2门设定，使用单一颜色控件，设置一个门包括使用FSA-A的散点图与线性缩放和通道的幅度只活细胞用于检测的L / D与污渍日志缩放。如:L / D阴性细胞。 </li><li>从门在4.1.3设置，使用单一的色彩控制，采用FSA-A的散点图与用于检测荧光蛋白细胞标记物与日志缩放通道的线性缩放和幅度设置一个门只包括活细胞阳性荧光。包括阳性细胞。 </li><li>设置任何补偿控制，如果需要的话，要占的L / D染色和荧光蛋白的光谱之间的干扰。 </li></ol></li><li>设置排序逻辑落入所有步骤设置4.1城门细胞。 <ol><li>采用双标记细胞，确认选通细胞分选。 注意:用于排序将细胞而不是碎屑，单个细胞而不是多聚L / D阴性和荧光阳性细胞的细胞。 </li></ol></li><li>排序2,000-4,000细胞从感兴趣的人口到在冰上的离心管5微升冷的FACS缓冲液中。为了在排序过程中尽量减少对细胞的剪切应变，用最低的压力可能使细胞分拣机。 注:FACS缓冲液的5微升液滴用作退出FACS机器的细胞的缓冲。收集2,000-4,000细胞直接进入FACS缓冲液消除装载到IFC芯片之前需要进行离心分离。建议采用此策略，因为离心可导致多聚形成的，如果细胞坚持颗粒彼此。 </li><li>评估排序后的可行性分析。 <ol><li>转移1微升排序细胞与100微升FACS分类缓冲器和1个新鲜FACS管:1000稀释的L / D污点。通过FACS分拣机与步骤4.1浇注战略传递细胞。使用L的比例/ D负到正来估计分选的细胞的生存力。 </li></ol></li></ol> 5.称重传感器到微流控芯片<ol><li>使用血细胞计数器，测量的浓度和分选的细胞的尺寸。 <ol><li>稀释排序细胞至少10微升。稀释5μ的等分试样; L细胞与5微升台盼蓝。加载到血球和应用盖玻片。 </li><li>收集所有细胞的亮场图像，4×4格血细胞计数。计数活细胞的数量，并计算活细胞/ ml。活细胞不占用台盼蓝。 注意:使用任何标准的图像分析软件来测量所有活细胞的直径。计算小区大小的平均值和标准偏差，并选择适合于感兴趣的细胞尺寸范围内的IFC板。如果单元格大小范围横跨国际金融公司板单元尺寸范围内，使用多个板来捕捉全方位的利益细胞。 </li></ol></li><li>上根据制造商的说明IFC板负载细胞（见材料）。 <ol><li>稀释细胞至1×10 6个细胞/ ml，并执行浮力优化按照制造商的说明。 注:浮力优化确保细胞既不沉底也不漂浮到装载WEL的顶L为取得最佳装载到IFC芯片。缓冲器的细胞的比例可以由细胞类型而有所不同，但通常为6:4-7:3细胞:缓冲器。 </li><li>加入细胞涂上底漆，IFC板。压板到兼容的流体机械，并运行一个小区负载脚本通过微流体电路进入采集通道，推动细胞。 </li></ol></li><li>确认细胞在国际金融公司板捕获网站提出。 <ol><li>卸下流体机械IFC板。安装在配备有板适配器显微镜的IFC板。 </li><li>在放大10倍收集亮场和荧光每次捕捉网站的快照。 注:明场捕获时间可能从10-50毫秒，这取决于灯泡的强度。荧光捕获时间可能从250-750毫秒，这取决于荧光亮度。以免过度曝光细胞光损伤预防。 </li></ol></li></ol> 6. cDNA合成<ol><li>根据我在原地进行细胞裂解FC板制造商的说明。 <ol><li>加入裂解缓冲液对IFC板的孔中。压板上兼容的流体机械及运行细胞裂解脚本按照制造商的说明。 </li></ol></li><li>根据国际金融公司板制造商的说明书进行反转录。 <ol><li>加入反转录试剂IFC板。压板上兼容的流体机械。运行逆转录脚本。样品循环条件:在25℃1个循环10分钟，在42℃下1个周期为1小时，然后在1周期在85℃下5分钟。 </li></ol></li><li>根据国际金融公司板制造商的说明用基因特异性探针进行前置放大。 注:由于他们与低拷贝数更大的特异性，采用荧光标记探针，而不是用于嵌入染料的使用最优化的探头。 <ol><li>池引物和稀释到每个最终180纳米。汇集引物添加到IFC板。压板上兼容fluidiCS机。前置放大运行脚本。 </li></ol></li><li>在95℃下在95℃变性15秒，然后退火和延伸在60℃下4分钟1个循环10分钟，然后18个循环:用下面的循环条件进行前置放大。在4℃直至收获商店预扩增的cDNA。嘉实cDNA的根据，直到使用制造商的说明和储存在-20℃微板。 </li></ol> 7.从单目标定量RT-PCR单细胞基因选择<ol><li>根据制造商的说明在25微升稀释试剂稀释的cDNA。 注:近似最终产量为28微升每单元前置放大cDNA的。保留稀释剂的等分试样用作定量RT-PCR检测的无模板对照。 <ol><li>谈到明场，并记录在步骤5.3.2荧光图像，得分每个捕获点。手动计数并记录细胞的数目。评估和记录每一个细胞是否感orescent。 注:每个单独的网站可能包含0，1，或&gt; 1的细胞，其中&gt; 1单元包括包含多个细胞和/或无法识别的碎片捕获点。如果图像是足够的质量，像素值也可以被分配给量化荧光信号的强度。 </li></ol></li><li>对于定量RT-PCR分析选择样本。 <ol><li>从步骤7.2确定了包含具有积极的荧光确切1细胞捕获地点选择cDNA样品。从每个样品的cDNA的池1微升等份。 注意:这是用来确定是否感兴趣的基因中的任何选定单元格中表达了"池"阳性对照。 </li><li>从包含恰好0细胞作为阴性对照的至少一个捕捉现场选择的cDNA。步骤7.1.2为无模板控制使用稀释剂。 </li></ol></li><li>运行定量RT-PCR检测。 <ol><li>仅使用在步骤6.4.1用于预扩增探针。加载triplicat所有样品即1个循环50℃2分钟，1个循环95℃，10分钟，40个循环，在95℃下15秒的变性，然后退火和延伸，在60℃进行1:用于在384孔板10微升反应循环条件分钟。 </li></ol></li></ol> 8.数据分析<ol><li>验证由标准凝胶电泳定量RT-PCR产物。确认产物是预期的分子量的单一条带（每个探头而异）。 注:如果探测器在不适当的大小产生多个频段或单波段，那么专一是值得怀疑的，这从排除分析基因。 </li><li>检查有无异常14所有扩增曲线和帐号。计算每个样品/基因组合15的平均CT值。 注:阳性对照CT值通常从10-30不等。为阴性对照CT值范围通常为35-40（无放大）。 CT值30-35被认为是非常低的表达应谨慎14来解释。 </li><li>报告数据<ol><li>如果样品倒在CT = 10-30，数据也可以被报告为相对转录丰度对照样品倍数变化。 注:折叠变化等于2 ^（ - ΔΔCT）其中ΔCT是相对于管家基因通过从管家基因减去样品的平均CT和ΔΔCT是样品和阳性对照15之间在ΔCT的差异。 </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+early+development+of+zebrafish.">Genetics and early development of zebrafish.</a> Trends in genetics : TIG. 5, 283-288 (1989).</li><li>Liu, J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+in+the+study+of+early+cardiac+development.">Zebrafish in the study of early cardiac development.</a> Circulation research. 110, 870-874 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. 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R., Land, H., Almudevar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+non-detects+in+qPCR+data.">On non-detects in qPCR data.</a> Bioinformatics. 30, 2310-2316 (2014).</li><li>Schmittgen, T. D., Livak, K. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+tinman+homolog+demarcates+the+heart+field+and+initiates+myocardial+differentiation.">Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation.</a> Development. 122, 3809-3816 (1996).</li><li>Zhou, Y., 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=Latent+TGF-beta+binding+protein+3+identifies+a+second+heart+field+in+zebrafish.">Latent TGF-beta binding protein 3 identifies a second heart field in zebrafish.</a> Nature. 474, 645-648 (2011).</li><li>Paffett-Lugassy, N., 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=Heart+field+origin+of+great+vessel+precursors+relies+on+nkx2.5-mediated+vasculogenesis.">Heart field origin of great vessel precursors relies on nkx2.5-mediated vasculogenesis.</a> Nature cell biology. 15, 1362-1369 (2013).</li><li>McCurley, A. T., Callard, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+housekeeping+genes+in+zebrafish:+male-female+differences+and+effects+of+tissue+type,+developmental+stage+and+chemical+treatment.">Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment.</a> BMC molecular biology. 9, 102 (2008).</li><li>Tang, R., Dodd, A., Lai, D., McNabb, W. C., Love, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+zebrafish+(Danio+rerio)+reference+genes+for+quantitative+real-time+RT-PCR+normalization.">Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization.</a> Acta biochimica et biophysica Sinica. 39, 384-390 (2007).</li><li>Serbedzija, G. N., Chen, J. N., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+in+the+heart+field+of+zebrafish.">Regulation in the heart field of zebrafish.</a> Development. 125, 1095-1101 (1998).</li><li>de Pater, E., 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=Distinct+phases+of+cardiomyocyte+differentiation+regulate+growth+of+the+zebrafish+heart.">Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart.</a> Development. 136, 1633-1641 (2009).</li><li>Hami, D., Grimes, A. C., Tsai, H. J., Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+cardiac+development+requires+a+conserved+secondary+heart+field.">Zebrafish cardiac development requires a conserved secondary heart field.</a> Development. 138, 2389-2398 (2011).</li><li>Yelon, D., Horne, S. A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+expression+of+cardiac+myosin+genes+reveals+regulated+aspects+of+heart+tube+assembly+in+zebrafish.">Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.</a> Developmental biology. 214, 23-37 (1999).</li><li>Molina, N., 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=Stimulus-induced+modulation+of+transcriptional+bursting+in+a+single+mammalian+gene.">Stimulus-induced modulation of transcriptional bursting in a single mammalian gene.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 20563-20568 (2013).</li></ol>

The authors have nothing to disclose.

本文所描述的方法使用荧光蛋白的表达下的细胞类型特异性启动子的控制，以丰富从斑马鱼胚胎中的微流体辅助单细胞捕获系统中使用的心脏祖细胞群，以评估在单个心动基因的一个子集的表达细胞。只要FACS激光激发和发射能力与所选择的荧光团（多个）相容，可以使用任何荧光报道线路此方法。许多斑马鱼记者线已经存在，并且可以在少至3个月后将产生携带新颖记者转基因鱼。此外，此工作?...

细胞和分子生物学目前大多数研究是基于人群的平均水平。然而，重要的生物事件可能由这些传统的以人群为基础的分析被屏蔽，因为较小的群体可以在生物过程和疾病预后扮演着重要的角色。理解在不同的人群的基因表达在单细胞水平可以（有）导致相关的生物和临床见解1,2。关注胚胎发育研究，在细胞的人口较多，祖细胞往往代表性不足，使得它具有挑战性的检测基因表达微妙的变化，最终引发细胞命运决定3。同样地，一个单一的细胞类型可响应于微环境4具有不同的表达谱。例如，在相同的器官或在不同的器官（ 例如 ，主动脉或肾）驻地内皮细胞表现显著异质尽管共享共同morphological和功能特点5。此外，癌细胞填充相同的肿瘤还可以在单细胞水平6不同分子型材或突变。 在模型系统中，单个细胞转录组已成功确定了新的细胞群，其特征细胞分化过程中发生的中间状态，并透露差细胞对刺激7,8,9。这样的见解会被掩盖了传统的基于人口的研究。斑马鱼的胚胎是干，祖的极大充分利用的源代码，并在开发过程中探索单细胞的异质性和细胞身份的分子调控问题分化细胞。他们的高度千篇一律， 体外发育和易于遗传操作使其成为这种方法10,11极好的模型系统。具体而言，主要的限制单细胞根的解释Ë表达数据是在开发过程中新的中间细胞状态的可靠的鉴定需要组织收集9非常小心时机。这是必要的，以确保捕获的细胞之间的异质性表示在单个时间点的组织内的异质性，而不是异质由年龄依赖性细胞分化呈现的基因表达。相比小鼠，斑马鱼胚胎发育可以精确在大量的胚胎12的同步。此外，具有大的离合器的尺寸，斑马鱼的胚胎可以用作干细胞和祖细胞的丰富来源。 这个协议描述来隔离从斑马鱼胚胎细胞和使用用于QRT-PCR的基因表达分析的市售集成微流体回路（IFC）芯片和Autoprep设备系统捕获单细胞的方法。该协议可迅速移植到任何高通量测定复包括全转录组测序，使细胞的异质性13的更全面的分析。它还提供了多种优势，传统的基因表达分析。单细胞分离协议产生的FACS后高存活率，这降低了包含在下游应用中受损细胞的比例。通过使用IFC，捕获细胞可以直接观察来评估捕获速率和形态学评估细胞的健康。此外，该协议是广泛适用于斑马鱼研究界，只需要一个标有转基因鱼线，并获得微流体细胞捕获技术。 作为一个原则的证明，从心脏祖细胞衍生的单细胞分离和IFC芯片上捕获，然后心脏分化标志物的相对丰度被定量RT-PCR检测。在单细胞水平的基因表达分析表明，心脏祖细胞与各色共存ntiating后代。从心脏祖细胞的单细胞分析的洞察力可以在脊椎动物的发育过程中心脏祖细胞中的基因表达模式的异质性，这可能在传统的基于人群的分析被掩盖线索。

<table><tbody><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>For removing the chorion from embryos</td></tr><tr><td>Microcentrifuge tube 2 ml</td><td>GeneMate</td><td>C-3261-1</td><td></td></tr><tr><td>40 um cell strainer</td><td>Biobasic</td><td>SP104151</td><td></td></tr><tr><td>35 mm culture dish</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>FACS tubes topped with 35 um cell strainer</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>P1000 and tips</td><td>Rainin</td><td>17005089</td><td></td></tr><tr><td>P20 and tips</td><td>Rainin</td><td>17005091</td><td></td></tr><tr><td>IFC chip manufacturer&#039;s protocol</td><td>Fluidigm</td><td>100-6117</td><td>Version 100-6117 E1 was used in representative experiment</td></tr><tr><td>Wide bore pastuer glass pippette</td><td>VWR</td><td>14673-010</td><td>For transferring embryos</td></tr><tr><td>Adult wild type zebrafish</td><td>N/A</td><td></td><td>We used AB line</td></tr><tr><td>Adult transgenic zebrafish</td><td>N/A</td><td></td><td>We used Tg(nkx2.5:ZsYellow)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for cell dissociation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Double distilled water</td><td>N/A</td><td></td><td></td></tr><tr><td>Instant Ocean Sea Salt</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>NaCl</td><td>FisherScientific</td><td>S271-3</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P5405</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>NaHCO3</td><td>Sigma Aldrich</td><td>S6014</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>Leibovitz&#039;s L-15</td><td>Gibco</td><td>21083-027</td><td></td></tr><tr><td>FBS</td><td>FisherScientific</td><td>03-600-511</td><td>Heat inactivate; any brand of FBS should be fine</td></tr><tr><td>Cell Dissociation Reagent 1 -TrypLE</td><td>Life Technologies</td><td>12605-010</td><td>Store at room temperature.</td></tr><tr><td>Cell Dissociation Reagent 2 - FACSmax</td><td>Genlantis</td><td>T200100</td><td>Store -20; thaw on ice; bring to room temp before use</td></tr><tr><td>pronase (optional)</td><td>Sigma</td><td>P5147</td><td></td></tr><tr><td>L/D Dye - Sytox Blue</td><td>Life Technologies</td><td>S34857</td><td>Any live/dead stain suitable for flow cytometry will work</td></tr><tr><td>Trypan Blue</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for IFC plate use and qRT-PCR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gene-specific probes</td><td></td><td></td><td>Probes will vary by experiment</td></tr><tr><td>TaqMan Probe ef1a</td><td>Life Technologies</td><td>Dr03432748_m1</td><td></td></tr><tr><td>TaqMan Probe gata4</td><td>Life Technologies</td><td>Dr03443262_g1</td><td></td></tr><tr><td>TaqMan Probe nk2-5</td><td>Life Technologies</td><td>Dr03074126_m1</td><td></td></tr><tr><td>TaqMan Probe myl7</td><td>Life Technologies</td><td>Dr03105700_m1</td><td></td></tr><tr><td>TaqMan Probe vmhc</td><td>Life Technologies</td><td>Dr03431136_m1</td><td></td></tr><tr><td>TaqMan Probe isl1</td><td>Life Technologies</td><td>Dr03425734_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Life Technologies</td><td>4369016</td><td></td></tr><tr><td>Reagents listed in IFC manufacturer&#039;s protocol</td><td></td><td></td><td></td></tr><tr><td>C1 Reagent Kit</td><td>Fluidigm</td><td>100-5319</td><td>Reagents for loading cells onto IFC plate</td></tr><tr><td>Ambion Single Cell-to-CT&lt;sup&gt;TM&lt;/sup&gt;</td><td>Life Technologies</td><td>4458237</td><td>Reagents for reverse transcription and pre-amplification steps</td></tr><tr><td>Molecular Biology Quality Water</td><td>Corning</td><td>46-000CM</td><td></td></tr><tr><td>C1 IFC for PreAmp (5-10 um)</td><td>Fluidigm</td><td>100-5757</td><td>IFC plate for small cells</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C1 AutoPrep machine</td><td>Fluidigm</td><td>100-5477</td><td>For IFC plate use</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Sigma Aldrich</td><td>Z359629</td><td>For counting cells and assessing cell size</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>For removing embryos from chorion</td></tr><tr><td>Tissue culture microscope</td><td></td><td></td><td>For assessing single cell digestion</td></tr><tr><td>FACS machine</td><td></td><td></td><td>For isolating cells of interest</td></tr></tbody></table>

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.

作为一个原则的证明，基因表达进行了评估心脏发育过程中，探索差异化的动态。在斑马鱼，心脏祖细胞从迁移到前侧板中胚层，他们融合形成线性心脏管细胞的中胚层人口出现。之前的融合，心脏祖细胞开始表达转录因子NKX2.5（NK 2同源5），它被认为是心脏祖16,17的最早特异性标记。在这里，先前描述的BAC转基因鱼的Tg（NKX2.5:ZsYellow）18，...

Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

Isolation and Characterization of Single Cells from Zebrafish Embryos

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 ...

isolation-and-characterization-of-single-cells-from-zebrafish-embryos

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Developmental Biology

14.5K 次观看.  University of North Carolina at Chapel Hill. 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. 

视频: Isolation and Characterization of Single Cells from Zebrafish Embryos

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

遗传学

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

发育生物学

Preparation of Single-Somite Explants from Zebrafish Embryos

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

We present a protocol for isolating single somites from zebrafish embryos, the dynamics of which can be followed in culture for several hours by fluorescence time-lapse microscopy, thus providing a methodology to quantify tissue-scale shape changes at single-cell resolution.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular ...

处理斑马鱼幼虫的肠道细胞以进行单细胞 RNA 测序

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

作者聚焦:分离和分析斑马鱼幼虫的肠道细胞以研究胃肠道发育的转录组学方面 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解析度全山<em&gt;在原位</em杂交斑马鱼胚胎内，研究基因表达和功能

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

分离和斑马鱼胚胎单细胞的表征

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此视频中的章节

在斑马鱼后脑细胞标记和成像

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

使用斑马鱼作为体内模型系统分析 DNA 复制定时

现场共聚焦显微镜对斑马鱼的胚胎脑成像

斑马鱼原代细胞的培养与转染

Preparation of Single-Somite Explants from Zebrafish Embryos

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

从斑马鱼幼虫中分离肠道进行单细胞RNA测序

高解析度全山