资助由国立卫生研究院 (NIH: K01-DK087814-01A1 到 D.L.S.), 加利福尼亚州立大学生物技术教育 & 研究计划 (CSUPERB: 脊椎动物造血龛的分子控制到 D.L.S.)来自加州州立大学的研究生研究办公室 (A.C.B.)。

加州州立大学动物保育和使用委员会 (IACUC) 咨询委员会批准了下文所述的所有方法。
1. 漂白 48 hpf 斑马鱼胚胎
<ol><li>与 15 cm (w) x 15 cm (l) x 7 cm (h) 塑料容器创造3洗站: 1) 与1毫升的5% 漂白在1000年毫升胚培养基 (E3; 见表 1), 2) 具无菌 E3 和 3) 具无菌 E3。确保每个容器有500毫升的溶液淹没胚胎。 注意: 对于每个测试的条件, 至少需要10胚胎。这种洗涤程序可容纳多达200胚胎。</li>
	<li>用转移吸管将胚胎放进茶叶过滤器中, 在洗涤站 #1 浸泡蛋5分钟, 取出茶叶滤网 (内胚), 放入洗涤站 #2 5 分钟;重复洗涤站 #3 为最后5分钟。</li>
	<li>最后一次洗涤后, 将其从茶叶过滤器中的胚胎翻转过来, 将其倒置到干净的 10 cm 培养皿中, 然后用无菌 E3 和转移吸管轻轻冲洗茶叶滤网的胚。</li>
</ol>2. Dechorionate 胚胎
<ol><li>用转移吸管, 去除和丢弃尽可能多的 E3 从培养皿和添加500μ l dechorionation 蛋白酶 (10 毫克/毫升) 的胚胎。室温孵育5分钟, 轻轻拍打培养皿的一侧, 完全去除 chorions。</li>
	<li>用一个血清吸管, 加入20毫升的无菌 E3 来稀释蛋白酶。允许胚胎沉淀, 用转移吸管移除 E3。重复这个洗涤步骤3次, 以消除所有的 dechorionation 蛋白酶的痕迹。</li>
</ol>3. 准备胚胎标本
<ol><li>使用 P1000 吸管, 将10胚胎放入单一的无菌1.5 毫升离心管。</li>
	<li>用吸管除去 E3 并丢弃。 注意事项: 移液管作为胚胎, 可以很容易地在下列洗涤步骤中移位和丢弃。</li>
</ol>4. 准备分离的胚胎
<ol><li>将样品转移到层流罩, 并通过加入1毫升无菌 E3 清洗胚胎。允许胚胎沉淀到管子底部, 用 P1000 吸管除去上清液。重复共3洗。</li>
	<li>最后一次洗涤后, 删除 E3 和丢弃。添加1毫升的10毫米糖 (E3), 以去除粘液涂层 (和任何孢子, 酵母, 或可能被困在它的细菌) 周围的胚胎斑马鱼。水平放置离心管, 室温下在层流罩内孵育25分钟。</li>
</ol>5. 胚胎分离
<ol><li>洗涤样品3次与1毫升无菌 DPBS (与 Ca2 +和 Mg2 +)。最后一次洗涤后添加500μ l DPBS (与 Ca2 +和 Mg2 +), 并添加5μ l 5 毫克/毫升 (26 U/毫升) 离解蛋白酶。 注意: 此酶需要 Ca2 +和 Mg2 +, 以确保 DPBS 含有这些离子。</li>
	<li>在 180 rpm 的水平轨道振动筛上孵育37° c 的样品, 在层流罩和研制胚中进行60分钟的取样, P1000 直到样品完全分离。 注意: 定期检查胚胎以确定它们何时分离。胚液应有一定数量的组织存在;它不应该是完全同源的。它是可能的过度消化, 将毁坏 HSPCs (参见补充图 1)。</li>
</ol>6. 游离胚胎的制备
<ol><li>将10分离的胚胎移到5毫升聚苯乙烯圆底管的顶部, 有35μ m 的细胞过滤器盖。</li>
	<li>使用吸管, 用无菌 PBS 冲洗培养管 (没有 Ca2 +或 Mg2 +), 并将解决方案传递给5毫升聚苯乙烯管, 其中含有6.1 步的过滤细胞。重复直到4毫升的液体是存在于5毫升聚苯乙烯管。 注意: 在这个阶段, 只要不包含 Ca2 +或 Mg2 + , 就可以使用 PBS、DPBS 或其他等等等渗缓冲解决方案。
</li>
	<li>离心管在4° c 和 300 x g 为 5 min 粒度均匀细胞样品。用吸管, 从圆底管上取出并丢弃上清液。注意不要扰乱在试管底部的细胞颗粒。重100μ l PBS 中的细胞。</li>
</ol>7. 纤维素的制备
<ol><li>生成1x 完整纤维素 (表 1) 库存解决方案, 并在3毫升注射器和 16 G 针的无菌圆底14毫升管中添加2.5 毫升。对每个测试的条件使用一个管。</li>
	<li>
添加细胞因子, 小分子, 或其他代理, 以调查每个样本。
	<ol>
<li>为髓质分化, 添加1% 鲤血清和0.3 毫克/毫升重组斑马鱼粒细胞集落刺激因子 (Gcsf)34到纤维素培养基。请参见博et al.37以充分说明如何生成鲤鱼血清和重组细胞因子。</li>
		<li>为红分化, 加入1% 鲤血清和0.1 毫克/毫升重组斑马鱼促红细胞生成素 (Epo)38纤维素培养基。</li>
		<li>要检查多向祖, 添加 Epo 和 Gcsf。 注意: 细胞因子和添加剂不应总体积的10% 以上, 因为培养基不会粘性足以辨别个体的菌落。</li>
	</ol>
</li>
</ol>8. 将游离胚胎加入纤维素
<ol><li>使用吸管, 添加100μ l 分离的10胚胎到表面的准备纤维素与重组细胞因子和鲤鱼血清。盖子和涡旋轻轻地完全质样品。每管现在应该包含10胚胎的游离组织。</li>
	<li>使用3毫升注射器和16克针, 分1毫升的样本分为2独立的 35 mm 培养皿。确保样品完全分散在培养皿中。对每个示例重复此操作。</li>
	<li>将 35 mm 培养皿中的细胞纤维素成15厘米的培养皿。每15厘米的菜, 添加一个35毫米的 Petri 板与5毫升的无菌水 humidify 样品。孵育在32° c 和 5% CO2为 7-10 天。</li>
</ol>9. HSPC 源集落形成单元 (CFUs) 的可视化
<ol><li>在孵化7天后, 将样品放置在倒置显微镜下, 在 40-100X 处进行可视化和计数。在这一点上, 个别的殖民地可以与吸管分离, 并受到染色和/或基因分析。</li>
</ol>

<ol>
	<li>Kondo, M., Weissman, I. L., Akashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clonogenic+common+lymphoid+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common lymphoid progenitors in mouse bone marrow.</a> Cell. 91, 661-672 (1997).</li><li>Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+common+myeloid+progenitor+that+gives+rise+to+all+myeloid+lineages.">A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.</a> Nature. 404, 193-197 (2000).</li><li>Bertrand, J. 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=Haematopoietic+stem+cells+derive+directly+from+aortic+endothelium+during+development.">Haematopoietic stem cells derive directly from aortic endothelium during development.</a> Nature. 464, 108-111 (2010).</li><li>Kissa, K., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+stem+cells+emerge+from+aortic+endothelium+by+a+novel+type+of+cell+transition.">Blood stem cells emerge from aortic endothelium by a novel type of cell transition.</a> Nature. 464, 112-115 (2010).</li><li>Bertrand, J. Y., Kim, A. D., Teng, S., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD41++cmyb++precursors+colonize+the+zebrafish+pronephros+by+a+novel+migration+route+to+initiate+adult+hematopoiesis.">CD41+ cmyb+ precursors colonize the zebrafish pronephros by a novel migration route to initiate adult hematopoiesis.</a> Development. 135, 1853-1862 (2008).</li><li>Bertrand, J. 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=Definitive+hematopoiesis+initiates+through+a+committed+erythromyeloid+progenitor+in+the+zebrafish+embryo.">Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo.</a> Development. 134, 4147-4156 (2007).</li><li>Ma, D., Zhang, J., Lin, H. F., Italiano, J., Handin, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+and+characterization+of+zebrafish+hematopoietic+stem+cells.">The identification and characterization of zebrafish hematopoietic stem cells.</a> Blood. 118, 289-297 (2011).</li><li>Lam, E. Y., Hall, C. J., Crosier, P. S., Crosier, K. E., Flores, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Runx1+expression+in+the+dorsal+aorta+tracks+the+emergence+of+blood+progenitors+from+endothelial+cells.">Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells.</a> Blood. 116, 909-914 (2010).</li><li>Lam, E. 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=Zebrafish+runx1+promoter-EGFP+transgenics+mark+discrete+sites+of+definitive+blood+progenitors.">Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors.</a> Blood. 113, 1241-1249 (2009).</li><li>Lin, H. F., 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=Analysis+of+thrombocyte+development+in+CD41-GFP+transgenic+zebrafish.">Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.</a> Blood. 106, 3803-3810 (2005).</li><li>Driever, W., 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=A+genetic+screen+for+mutations+affecting+embryogenesis+in+zebrafish.">A genetic screen for mutations affecting embryogenesis in zebrafish.</a> Development. 123, 37-46 (1996).</li><li>Weinstein, B. M., 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=Hematopoietic+mutations+in+the+zebrafish.">Hematopoietic mutations in the zebrafish.</a> Development. 123, 303-309 (1996).</li><li>Ransom, D. G., 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=Characterization+of+zebrafish+mutants+with+defects+in+embryonic+hematopoiesis.">Characterization of zebrafish mutants with defects in embryonic hematopoiesis.</a> Development. 123, 311-319 (1996).</li><li>Amsterdam, 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=A+large-scale+insertional+mutagenesis+screen+in+zebrafish.">A large-scale insertional mutagenesis screen in zebrafish.</a> Genes Dev. 13, 2713-2724 (1999).</li><li>Gaiano, 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=Insertional+mutagenesis+and+rapid+cloning+of+essential+genes+in+zebrafish.">Insertional mutagenesis and rapid cloning of essential genes in zebrafish.</a> Nature. 383, 829-832 (1996).</li><li>Yeh, J. R., 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=Discovering+chemical+modifiers+of+oncogene-regulated+hematopoietic+differentiation.">Discovering chemical modifiers of oncogene-regulated hematopoietic differentiation.</a> Nat Chem Biol. 5, 236-243 (2009).</li><li>Paik, E. J., de Jong, J. L., Pugach, E., Opara, P., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+genetic+screen+in+zebrafish+for+pathways+interacting+with+cdx4+in+primitive+hematopoiesis.">A chemical genetic screen in zebrafish for pathways interacting with cdx4 in primitive hematopoiesis.</a> Zebrafish. 7, 61-68 (2010).</li><li>Ridges, 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=Zebrafish+screen+identifies+novel+compound+with+selective+toxicity+against+leukemia.">Zebrafish screen identifies novel compound with selective toxicity against leukemia.</a> Blood. 119, 5621-5631 (2012).</li><li>North, T. 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=Prostaglandin+E2+regulates+vertebrate+haematopoietic+stem+cell+homeostasis.">Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis.</a> Nature. 447, 1007-1011 (2007).</li><li>Astuti, 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=A+Functional+Bioluminescent+Zebrafish+Screen+for+Enhancing+Hematopoietic+Cell+Homing.">A Functional Bioluminescent Zebrafish Screen for Enhancing Hematopoietic Cell Homing.</a> Stem Cell Reports. 8, 177-190 (2017).</li><li>Kiel, M. 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=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li><li>Spangrude, G. J., Heimfeld, S., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+mouse+hematopoietic+stem+cells.">Purification and characterization of mouse hematopoietic stem cells.</a> Science. 241, 58-62 (1988).</li><li>Morrison, S. J., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+repopulating+subset+of+hematopoietic+stem+cells+is+deterministic+and+isolatable+by+phenotype.">The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.</a> Immunity. 1, 661-673 (1994).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273, 242-245 (1996).</li><li>Goodell, M. 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=Dye+efflux+studies+suggest+that+hematopoietic+stem+cells+expressing+low+or+undetectable+levels+of+CD34+antigen+exist+in+multiple+species.">Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.</a> Nat Med. 3, 1337-1345 (1997).</li><li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183, 1797-1806 (1996).</li><li>Gansner, J. M., 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=Sorting+zebrafish+thrombocyte+lineage+cells+with+a+Cd41+monoclonal+antibody+enriches+hematopoietic+stem+cell+activity.">Sorting zebrafish thrombocyte lineage cells with a Cd41 monoclonal antibody enriches hematopoietic stem cell activity.</a> Blood. 129, 1394-1397 (2017).</li><li>Smith, A. 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=High-throughput+cell+transplantation+establishes+that+tumor-initiating+cells+are+abundant+in+zebrafish+T-cell+acute+lymphoblastic+leukemia.">High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia.</a> Blood. 115, 3296-3303 (2010).</li><li>Mizgirev, I., Revskoy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+clonal+zebrafish+lines+and+transplantable+hepatic+tumors.">Generation of clonal zebrafish lines and transplantable hepatic tumors.</a> Nat Protoc. 5, 383-394 (2010).</li><li>de Jong, J. L., 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=Characterization+of+immune-matched+hematopoietic+transplantation+in+zebrafish.">Characterization of immune-matched hematopoietic transplantation in zebrafish.</a> Blood. 117, 4234-4242 (2011).</li><li>Wolf, 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=Zebrafish+Caudal+Haematopoietic+Embryonic+Stromal+Tissue+(CHEST)+Cells+Support+Haematopoiesis.">Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis.</a> Sci Rep. 7, 44644 (2017).</li><li>Campbell, 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=Zebrafish+embryonic+stromal+trunk+(ZEST)+cells+support+hematopoietic+stem+and+progenitor+cell+(HSPC)+proliferation,+survival,+and+differentiation.">Zebrafish embryonic stromal trunk (ZEST) cells support hematopoietic stem and progenitor cell (HSPC) proliferation, survival, and differentiation.</a> Exp Hematol. 43, 1047-1061 (2015).</li><li>Svoboda, O., 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=Dissection+of+vertebrate+hematopoiesis+using+zebrafish+thrombopoietin.">Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin.</a> Blood. 124, 220-228 (2014).</li><li>Stachura, D. L., 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=The+zebrafish+granulocyte+colony-stimulating+factors+(Gcsfs):+2+paralogous+cytokines+and+their+roles+in+hematopoietic+development+and+maintenance.">The zebrafish granulocyte colony-stimulating factors (Gcsfs): 2 paralogous cytokines and their roles in hematopoietic development and maintenance.</a> Blood. 122, 3918-3928 (2013).</li><li>Stachura, D. L., 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=Clonal+analysis+of+hematopoietic+progenitor+cells+in+the+zebrafish.">Clonal analysis of hematopoietic progenitor cells in the zebrafish.</a> Blood. 118, 1274-1282 (2011).</li><li>Stachura, D. L., 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=Zebrafish+kidney+stromal+cell+lines+support+multilineage+hematopoiesis.">Zebrafish kidney stromal cell lines support multilineage hematopoiesis.</a> Blood. 114, 279-289 (2009).</li><li>Svoboda, O., 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=Ex+vivo+tools+for+the+clonal+analysis+of+zebrafish+hematopoiesis.">Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.</a> Nat Protoc. 11, 1007-1020 (2016).</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=Functional+conservation+of+erythropoietin+signaling+in+zebrafish.">Functional conservation of erythropoietin signaling in zebrafish.</a> Blood. 110, 2718-2726 (2007).</li><li>Stachura, D. L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 133, 11-53 (2016).</li><li>Traver, D., 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat Immunol. 4, 1238-1246 (2003).</li><li>Kobayashi, I., 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=Characterization+and+localization+of+side+population+(SP)+cells+in+zebrafish+kidney+hematopoietic+tissue.">Characterization and localization of side population (SP) cells in zebrafish kidney hematopoietic tissue.</a> Blood. 111, 1131-1137 (2008).</li><li>Henninger, 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=Clonal+fate+mapping+quantifies+the+number+of+haematopoietic+stem+cells+that+arise+during+development.">Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development.</a> Nat Cell Biol. 19, 17-27 (2017).</li><li>Tamplin, O. 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=Hematopoietic+stem+cell+arrival+triggers+dynamic+remodeling+of+the+perivascular+niche.">Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.</a> Cell. 160, 241-252 (2015).</li></ol>

作者没有什么可透露的。

斑马鱼模型系统已成为研究原始和最终脊椎动物造血的有效、有效和廉价的模型。快速、廉价和目前很少的技术难题的产生可以用来测试小分子, 分析突变的胚胎, 以及阐明对 HSPC 生物学重要的分子通路。体外HSPCs 从成人斑马鱼中电镀是一种研究诱变、细胞因子和造血缺损的有效方法。这项协议的基础上, 这些研究, 但利用胚胎斑马鱼。将这些化验结果应用于胚胎斑马鱼, 可以更快地收集数据...

造血是使生物体存活所需的大量成熟血细胞的过程。这是一个关键的发展过程, 涉及分化的造血干细胞 (干细胞) 到各种发育限制细胞类型, 包括成熟的血液。这些干细胞必须自我更新, 使系统永远不会枯竭, 他们必须坚持从早期胚胎发育直至死亡。在脊椎动物中, 需要不断分化和增殖造血干和祖细胞 (HSPCs), 以充分补充大多数的血液细胞后有丝分裂和每天回收。干细胞先分化成限制性祖细胞的亚群, 生成成熟的血细胞;常见的淋巴祖细胞 (CLPs)1, 最终产生 T、B 和 NK 淋巴细胞, 以及常见的髓细胞祖 (中医)2 , 产生粒、红细胞、巨噬和巨。这些祖细胞致力于产生特定的细胞谱系, 并进一步分化为更发育的限制性前体细胞, 如髓红祖 (欧洲议员), 产生红细胞和血小板, 或粒细胞巨噬细胞祖 (gmp), 可产生嗜、嗜酸性粒细胞、嗜中性和巨噬细胞2。识别和隔离这些祖细胞允许识别重要的分子通路参与造血分化和许多造血疾病, 如白血病出现时, 这些前体细胞未能正确区分.
在过去的几十年中, 斑马鱼 (斑马鱼) 模型系统已经成为胚胎和成人造血研究的关键研究工具。斑马鱼是适于遗传分析, 是 phylogenetically 最低的脊椎动物模型物种, 有类似的血管和造血系统的人。斑马鱼胚胎发育前子宫,并在48小时后受精 (hpf) 生成 HSPCs3,4,5,67, 8。斑马鱼也高度肥沃, 雌性在一个单一的离合器中放置超过100鸡蛋, 允许大样本大小和实验复制。斑马鱼的胚胎是光学透明的, 允许对造血系统进行显微可视化。斑马鱼的一些荧光转基因线标记干细胞如runx1: EGFP 鱼类9、 cd41: EGFP 鱼10和kdrl:mCherry;cmyb: GFP3双阳性动物, 允许现场直播, 实时可视化 HSC 的出现和扩展在体内3,4,7,8, 9。斑马鱼的快速生成时间和发育前子宫导致其在诱变研究中的应用11,12,13,14,15和药物筛选16,17,18,19,20 , 用于对人类血液疾病有治疗前景的化合物。总的来说, 造血系统的保护, 转基因线的存在和容易的发展, 和快速的再生时间使斑马鱼成为一个廉价, 快速, 灵活, 理想的造血研究模式。
在哺乳动物的造血系统中已经开发出许多分离和检测干细胞的方法。调查人员可以利用细胞表面感受器的组合来标记干细胞21,22,23,24, 以及利用干细胞的能力来流出染料25,26。在它们被标记后, 荧光活化细胞分选 () 允许他们的物理分离。证明细胞是 HSC 需要照射宿主动物来破坏内源性 HSPCs, 移植假定的干细胞, 并观察所有成熟的血细胞类型的长期的多谱系重组。这些检测方法在小鼠中很好的工作, 因为有许多细胞表面抗体对造血细胞和近交系小鼠菌株, 促进免疫匹配的移植。然而, 很少有斑马鱼造血细胞表面抗体产生27, 阻碍了干细胞的识别和分离。最常见的方法来标记和孤立斑马鱼干细胞是与转基因动物, 藉以细胞特异启动子序列驾驶荧光蛋白的表达。研究在背主动脉腹壁上进行了可视化和枚举, 利用这一技术的显微镜干细胞3,4,8,9。其他实验室产生了斑马鱼的克隆菌株28,29 , 并在 MHC 匹配的动物中进行了成功的移植30。然而, 这些技术是成本高昂的许多实验室, 在技术上是困难的, 是费时的。为了解决这些问题, 实验室已经生成了几个体外化验, 以测试 HSPCs 的存在、增殖率和差异化能力31、32、33, 34、35、36。这些化验显示 HSPCs 的增殖和分化在体外31,32,33,34,35,36, 抢救造血缺损36, 以及一种有效的发现和检测细胞因子的方法33,34,35。它们也被用来鉴定负责 HSPC 生物学的基因31,32。在这项研究中, 我们进一步采取这些化验, 允许定量的 HSPCs 在发展中的斑马鱼胚胎。这些化验也可以用来定量的数量 HSPCs 突变动物和动物治疗的造血破坏性药物。从本质上讲, 这些化验是快速的, 目前很少的技术挑战, 是廉价的方法, 定量 HSPC 数字, 检查其增殖, 并调查块的分化。

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		<tr height="40">
			<td height="40" style="height:40px;width:341px;">10% Bovine Serum Albumin in Iscove&#39;s MDM</td>
			<td style="width:115px;">StemCell Technologies&#160;</td>
			<td style="width:119px;">9300</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">DPBS (10x) with Calcium2+ and Magnesium2+</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:119px;">14080-055</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">HyClone PBS (1x)</td>
			<td style="width:115px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">sh30256.01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">&#160;DMEM 500 mL</td>
			<td style="width:115px;">Corning CellGrow</td>
			<td style="width:119px;">10-017-CV</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">&#160;Ham&#39;s F12 500 mL</td>
			<td style="width:115px;">Corning CellGrow</td>
			<td style="width:119px;">10-080-CV</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">FBS 500 mL</td>
			<td style="width:115px;">Gemini Bio-Products</td>
			<td style="width:119px;">100-108</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">HEPES 100 mL (1 M)</td>
			<td style="width:115px;">Gibco life technologies</td>
			<td style="width:119px;">15-630-080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Penicillin/streptomycin (5000 U/mL 
			and 5000 mg/mL) with L-Glutamine (200 mM)</td>
			<td style="width:115px;">Corning Mediatech</td>
			<td style="width:119px;">30-009-CI</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Gentamycin Sulfate 10 mL (50 mg/mL)</td>
			<td style="width:115px;">Corning Mediatech</td>
			<td style="width:119px;">30-005-CR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">1.5 mL MCF tube</td>
			<td style="width:115px;">FisherBrand</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">3 mL 23gx1 injection needle with Luer lock</td>
			<td style="width:115px;">BD Safety Glide</td>
			<td style="width:119px;">305905</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">5 mL polystyrene round bottom tube with cell strainer cap</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">Methocel MC</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">64630</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">14 mL Polystyrene round bottom tube</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">352057</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">10 mm polystyrene easygrip Petri dish</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">351008</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Librease TM</td>
			<td style="width:115px;">Roche Sigma-Aldrich</td>
			<td>5401119001</td>
			<td>dissociation protease</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Pronase</td>
			<td style="width:115px;">Roche Sigma-Aldrich</td>
			<td style="width:119px;">11459643001</td>
			<td>dechorionation protease</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">15 cm Petri dish</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:341px;">AB</td>
			<td style="width:115px;">Zebrafish International Resource Center (ZIRC)</td>
			<td style="width:119px;">ZL1</td>
			<td>zebrafish strain used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">Dithiolthreitol (DTT)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos

造血是一种必需的细胞过程, 造血干细胞和祖细胞 (HSPCs) 分化成含有成熟血液的不同细胞谱系的众多。隔离和识别这些 HSPCs 很困难, 因为它们被定义为事后; 它们只能在分化成特定的细胞谱系后定义。在过去的几十年里, 斑马鱼 (斑马鱼) 已经成为研究造血的模范有机体。斑马鱼胚胎发育为宫内, 48 小时后受精 (hpf) 已经产生了明确的 HSPCs. 通过移植和随后的研究, 对 HSPC 分化和增殖能力进行了评估。重组的造血系统, 除了可视化专业的转基因线与共焦显微镜。然而, 这些化验是成本高昂, 技术上的困难, 和耗时的许多实验室。开发一个体外模型来评估 HSPCs 将是成本效益高, 速度更快, 并目前较少的困难相比, 先前所描述的方法, 允许实验室迅速评估突变和药物屏幕, 影响 HSPC 生物学。这本新的体外测定 HSPCs 的方法是通过电镀全斑马鱼胚胎和添加外源因素, 促进只有 HSPC 分化和增殖。胚胎被分离成单个细胞, 并镀有 HSPC 支持的集落刺激因子, 导致它们产生由单个祖细胞产生的菌落形成单元 (CFUs)。这些分析应允许更仔细地检查的分子通路, 负责 HSPC 增殖, 分化和调控, 这将使研究人员了解的基础脊椎动物造血和其失调在疾病期间。

为了评估 HSPC 的数量在胚胎斑马鱼, 48 hpf 胚胎被消化, 镀在纤维素与外源性造血支持生长因子, 并孵育7天 (图 1A)。7天后, 对菌落形成单元 (CFUs) 进行了枚举 (图 1B) 和映像 (图 1C)。通过控制不同的细胞因子增加, 你可以控制的类型的殖民地产生: Epo 是必要的红分化和 Gcsf ...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

在这里, 我们提出了一个简单的方法, 定量造血干细胞和祖细胞 (HSPCs) 在胚胎斑马鱼。HSPCs 从分离的斑马鱼被镀在纤维素与支持因素, 分化成成熟的血液。这使得检测血液缺陷和允许药物筛选很容易进行。

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

development-an-vitro-assay-to-quantitate-hematopoietic-stem

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

7.9K 次观看.  California State University, Chico. 在这里, 我们提出了一个简单的方法, 定量造血干细胞和祖细胞 (HSPCs) 在胚胎斑马鱼。HSPCs 从分离的斑马鱼被镀在纤维素与支持因素, 分化成成熟的血液。这使得检测血液缺陷和允许药物筛选很容易进行。

视频: Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

造血干细胞代谢分析

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

摘要

探索更多视频

此视频中的章节

人类神经干细胞从外周血造血祖细胞直接诱导

技术目标显微注射到显影

联体斑马鱼胚胎一代发展囊胚的手术融合

人多能干细胞对原始和最终造血祖细胞定向分化的研究

诱导生活青蛙和斑马鱼胚胎缺氧

活完好斑马鱼的单细胞 Photoconversion

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

造血干细胞代谢分析

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

摘要

探索更多视频

此视频中的章节

人类神经干细胞从外周血造血祖细胞直接诱导

技术目标显微注射到显影

联体斑马鱼胚胎一代发展囊胚的手术融合

人多能干细胞对原始和最终造血祖细胞定向分化的研究

诱导生活青蛙和斑马鱼胚胎缺氧

活完好斑马鱼的单细胞 Photoconversion

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

造血干细胞代谢分析

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应