Name: identification and analysis of mouse erythroid progenitors using the cd71/ter119 flow-cytometric assay
Uploaded: 2011-08-05T13:00:00
Description: Watch this Scientific Journal Video about Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay at JoVE.com

我们感谢麻省大学的流式细胞仪的核心是：理查德Konz，特德Giehl，芭芭拉Gosselin，粤华谷和塔米Krupoch。这项工作是由美国国立卫生研究院/ NHLBI RO1 HL084168（MS）和美国国立卫生研究院CA的T32 - 130807（JRS）。也可用于糖尿病内分泌研究中心授予DK32520支持的核心资源。

1。收获的组织<ol><li>准备管含有2至5毫升冷染色缓冲液（磷酸盐缓冲液（PBS）与添加0.2％BSA和5mm葡萄糖）。置于冰上管组织收获前。 </li><li>根据卡尔小鼠适当批准的协议（如CO 2吸入颈椎脱位）。 </li><li> EDTA或肝素血液收集管进入心脏穿刺，为以后的分析，如红细胞压积，网织红细胞计数或CBC分析，绘制血。 </li><li>收获脾和骨骼，放置在一个单独的管，准备在第1步，从每个鼠标的组织?...

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	<li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+Fas-FasL+coexpression+by+erythropoietin+mediates+erythroblast+expansion+during+the+erythropoietic+stress+response+in.">Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in.</a> 108, 123-133 (2006).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Autoregulation+by+FAS+Mediates+Robust+Fetal+Erythropoiesis.">Negative Autoregulation by FAS Mediates Robust Fetal Erythropoiesis.</a> PLoS Biol. 5, e252-e252 (2007).</li><li>Krutzik, P. O., Hale, M. B., Nolan, G. P. <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+the+murine+immunological+signaling+network+with+phosphospecific+flow+cytometry.">Characterization of the murine immunological signaling network with phosphospecific flow cytometry.</a> J Immunol. 175, 2366-2373 (2005).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ineffective+erythropoiesis+in+Stat5a(-/-)5b(-/-)+mice+due+to+decreased+survival+of+early+erythroblasts.">Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.</a> Blood. 98, 3261-3273 (2001).</li><li>Guihard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAPK+ERK1+is+a+negative+regulator+of+the+adult+steady-state+splenic+erythropoiesis.">The MAPK ERK1 is a negative regulator of the adult steady-state splenic erythropoiesis.</a> Blood. 115, 3686-3694 (2010).</li><li>Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+erythroid+chaperone+that+facilitates+folding+of+alpha-globin+subunits+for+hemoglobin+synthesis.">An erythroid chaperone that facilitates folding of alpha-globin subunits for hemoglobin synthesis.</a> J Clin Invest. 117, 1856-1865 (2007).</li><li>Chen, 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=Erythroid+dysplasia,+megaloblastic+anemia,+and+impaired+lymphopoiesis+arising+from+mitochondrial+dysfunction.">Erythroid dysplasia, megaloblastic anemia, and impaired lymphopoiesis arising from mitochondrial dysfunction.</a> Blood. 114, 4045-4053 (2009).</li><li>Chen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+distinct+stages+in+erythroid+differentiation+based+on+dynamic+changes+in+membrane+protein+expression+during+erythropoiesis.">Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.</a> Proc Natl Acad Sci U S A. 106, 17413-17418 (2009).</li><li>McGrath, K. E., Bushnell, T. P., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+hematopoietic+cells:+where+flow+meets+morphology.">Multispectral imaging of hematopoietic cells: where flow meets morphology.</a> J Immunol Methods. 336, 91-97 (2008).</li><li>Pop, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+key+commitment+step+in+erythropoiesis+is+synchronized+with+the+cell+cycle+clock+through+mutual+inhibition+between+PU.1+and+S-phase+progression.">A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression.</a> PLoS Biol. 8, (2010).</li><li>Borsook, H., Lingrel, J. B., Scaro, J. L., Millette, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+haemoglobin+in+relation+to+the+maturation+of+erythroid+cells.">Synthesis of haemoglobin in relation to the maturation of erythroid cells.</a> Nature. 196, 347-350 (1962).</li></ol>

流流式细胞仪的方法允许同时调查任何可能与一个特定的荧光标记抗体或配体，包括细胞表面标志物，蛋白表达，细胞的存活，使用磷酸化特异性抗体和细胞周期状态的细胞信号检测的细胞功能。这些测量可以在每一个分化阶段的特定子集，在新鲜分离的红细胞生成组织范围内。因此，这种方法允许红细胞生成系统的不同层次的功能和分子的变化进行评估，在广泛的红细胞生成刺激或基因突变的?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> FAS -生物素</td><td> BD Pharmingen公司</td><td> 554256 </td></tr><tr><td>链霉亲和APC </td><td>分子探针</td><td> S868 </td></tr><tr><td> 40微米的无菌细胞过滤器</td><td> Fisherbrand </td><td> 22363547 </td></tr><tr><td>流式细胞仪染色聚苯乙烯圆底管</td><td>屋宇署猎鹰</td><td> 352008 </td></tr><tr><td> U型底96孔板</td><td>屋宇署猎鹰</td><td> 353910 </td></tr><tr><td> ChromePure兔IgG </td><td>杰克逊ImmunoResearch </td><td> 015-000-003 </td></tr><tr><td> CD71 - FITC（股票0.5mg/ml的） </td><td> BD -生物科学</td><td> 553266 </td></tr><tr><td> Ter119 - PE（股票0.2mg/ml） </td><td> BD -生物科学</td><td> 553673 </td></tr><tr><td> 7AAD </td><td> BD -生物科学</td><td> 559925 </td></tr><tr><td> DAPI染色粉</td><td>罗氏公司</td><td> 236276 </td></tr><tr><td> FITC标记的大鼠抗小鼠CD41 MWReg30 </td><td> BD Pharmingen公司</td><td> 553848 </td></tr><tr><td> FITC标记的大鼠抗小鼠CD45R/B220，RA3 - 6B2 </td><td> BD Pharmingen公司</td><td> 553087 </td></tr><tr><td> FITC标记的大鼠抗小鼠CD411b/Mac-1 M1/70 </td><td> BD Pharmingen公司</td><td> 557396 </td></tr><tr><td> FITC标记的鼠抗鼠LY - 6G和Ly - 6C（GR - 1）RB6 - 8C5 </td><td> BD Pharmingen公司</td><td> 553126 </td></tr><tr><td> FITC标记的鼠抗鼠CD3e 145 - 2C11 </td><td> BD Pharmingen公司</td><td> 553061 </td></tr><tr><td> APC BrdU流试剂盒</td><td> BD Pharmingen公司</td><td> 557892 </td></tr><tr><td> Annexin V的抗生物素蛋白</td><td> BD Pharmingen公司</td><td> 556418 </td></tr></tbody></table>

identification and analysis of mouse erythroid progenitors using the cd71/ter119 flow-cytometric assay

红细胞的研究，旨在了解红细胞是如何从早期的造血和红系祖细胞形成。具体来说，是受激素的促红细胞生成素（EPO），其合成是由组织缺氧引起红细胞的形成率。适当组织在迅速增加EPO的充氧效果，驾驶增加红细胞生成率的威胁，被称为红细胞生成应激反应的一个过程。在循环红细胞的数量增加，提高组织的氧输送。红细胞生成一个高效的应激反应，因此，从生理和病理条件下，如高海拔，贫血，出血，化疗或造血干细胞移植的生存和恢复的关键。 鼠标是红细胞和应激反应的研究重点型号。鼠标最终（成人型）红细胞在胚胎12.5和15.5之间天的胎肝，脾中的新生儿，在成人脾和骨髓。确定组织中的红系祖细胞的经典方法依赖于这些细胞的能力，引起红细胞殖民地时，在半固体培养基含有EPO -镀。他们的红易制毒化学后代基于形态学标准确定。无论这些经典的方法能够获得大量的细胞分子研究的特定分化阶段红。在这里，我们目前的流量识别和研究直接在新鲜分离的小鼠组织中的分化阶段特有的红系祖细胞和前体，流式细胞仪方法。检测依赖于细胞表面标志物CD71，Ter119，流流式细胞仪“向前散射”的参数，这是一个细胞大小的功能。 CD71/Ter119法可用于研究红系祖细胞在其体内红细胞生成应力的响应，例如，住在贫血小鼠或老鼠在低氧条件。直接转基因的成年小鼠或胚胎组织，它也可以用来研究红系祖细胞，修改后的红细胞中的分子途径，以评估的具体作用。

Watch this Scientific Journal Video about Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay at JoVE.com

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

一个流式细胞仪方法，鉴定和分化阶段特异性的小鼠红系祖细胞和前体，直接在新鲜收获的小鼠骨髓，脾脏或胎肝的分子分析流。检测依赖于细胞表面标志物CD71，Ter119，细胞大小。

使用CD71/TER119流流式细胞仪检测小鼠红系祖细胞的识别和分析

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