Name: identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry
Uploaded: 2017-07-17T12:30:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

作者感谢Frederic Geissmann教授和Christian Schulz教授进行了深入的讨论;汉娜·加纳博士对手稿的批判性阅读，Xavier Montagutelli博士和Jean Jaubert博士以及巴斯德研究所动物设施的工作人员，以支持畜牧业;以及他们的技术援助Pascal Dardenne和一名安进学者Vytaute Boreikaite。 EGP实验室的研究由巴斯德研究机构，CNRS，Cercle FSER（FRM和来自巴斯德研究所和REVIVE联盟的起始包）资助，LI由REVIVE联盟的博士研究生资助。

根据巴斯德研究所（CETEA）批准的机构动物护理和使用委员会进行动物手术。 1. 在 CSF1R MeriCreMer 罗莎 LSL-YFP胚胎子宫脉冲标记<ol><li> 准备4-羟基他莫昔芬（OH-TAM，50 mg / mL）的储备溶液。 <ol><li>在通风柜下，打开25毫克小瓶的OH-TAM，加入250μL乙醇（100％）。 </li><li>将OH-TAM溶液转移到具有截头的2mL微量离心管中，并以最大速度涡旋10分钟。 </li><li>在超声波浴中超声30分钟。 </li><li>在通风柜内加入250μL的PEG-35蓖麻油，得到50mg / mL的储备溶液。 注意:PEG-35蓖麻油是具有两亲性质的溶剂，其结合疏水性分子并将其溶解在含水溶剂中。 </li><li>漩涡约5分钟在超声波浴中以最大速度超声处理30分钟。 </li><li>每个微量离心管（每次注射1次）分装90μL（4.5mg）。 </li><li>储存于4℃下放置1周或在-20℃下长期先前描述的13。 </li></ol></li><li> 准备孕酮（10mg / mL） <ol><li>向25mg孕酮中加入250μL的100％乙醇以在通风柜下制备10mg /100μL悬浮液。轻轻旋转</li><li>加入2250μL高压消毒的向日葵油，在罩下制成10mg / ml孕酮溶液。 </li><li>以最大速度涡旋约5分钟，等分并储存在4°C。请注意，存储时解决方案是清晰的。 </li></ol></li><li> 他莫昔芬 注意:胚胎发育估计考虑阴道栓塞形成的日子为胚胎日（E）0.5。 E8.5单次注射诱导重组进入孕妇Csf1r MeriCreMer女性 12 。补充OH-TAM与37.5μg/ g孕酮，以降低他莫昔芬给药后的流产率。下午1点注射（早晨观察阴道塞）。 <ol><li>在注射的早晨，将OH-TAM溶液的等分试样超声处理10分钟（或直到完全重悬）。 </li><li>在通风柜内加入360μLNaCl 0.9％，得到10mg / mL OH-TAM溶液，并彻底涡旋。超声处理直到溶液澄清并完全重悬（至少30分钟）。 </li><li>预热孕酮至室温。 </li><li>在微量离心管中混合450μL的OH-TAM和225μL的孕酮。涡流。 </li><li>超声处理至少10分钟，并用25G针装入1 mL注射器。 </li><li>在动物设施中，称重怀孕的雌性（ Csf1r MeriCreMer女性处于近交的FVB遗传背景和典型的体重en 25和33g）。 </li><li>通过将计算出的体积缓慢注入鼠标来进行腹膜内注射。取出针后，轻轻按压穿刺伤口，按摩腹部分布OH-TAM。 注意:OH-TAM注射剂量为75 mg / kg，孕酮剂量为37.5 mg / kg。 表1提供注射到怀孕女性的体积。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>小鼠体重（g） </td><td>从混合物注射的总体积（μL） </td></tr><tr><td> 25 </td><td> 281 </td></tr><tr><td> 26 </td><td> 292 </td></tr><tr><td> 27 </td><td> 303 </td></tr><tr><td> 28 </td><td> 315 </td></tr><tr><td> 29 </td><td> 326 </td></tr><tr><td>三十</td><td> 338 </td></tr><tr><td> 31 </td><td> 348 </td></tr><tr><td> 32 </td><td> 360 </td> &lt;/ TR&gt; <tr><td> 33 </td><td> 371 </td></tr><tr><td> 34 </td><td> 382 </td></tr><tr><td> 35 </td><td> 394 </td></tr></tbody></table> 表1:4-OH-他莫昔芬（OH-TAM）的注射体积。单次注射的单次注射量为75μg/ g（体重）的补充有37.5μg/ g孕酮的OH-TAM。 2.卵黄囊（YS）和胎肝（FL）的解剖注意:严格的无菌技术在操作胚胎时是不必要的，除非它们将被用于长期培养。然而，工作区域必须清洁，并在吸收纸上覆盖箔。 <ol><li>制备冰冷的磷酸盐缓冲盐水（PBS）和消化混合物（含有1mg / mL胶原酶D，100U / mL脱氧核糖核酸酶I（DNase I）和3％胎牛血清的PBS）。 </li><li>牺牲怀孕的女性由cer在所需的妊娠日（ 例如胚胎阶段E10.5）的动脉脱位。 </li><li>将皮肤紧贴在生殖器上，并用剪刀在中线做一个小切口。将皮肤拉向头部，完全暴露身体，无毛皮。切开腹肌露出内脏，推动肠道露出两个子宫角。 </li><li>用中等大小的镊子抓住附着在卵巢上的脂肪垫，轻轻拉出子宫。在子宫颈的颈部切开并从腹腔抬起角。去除脂肪垫以完全释放子宫角，并从每侧的卵巢切割角。 </li><li>将角状物置于10毫米培养皿中的冰冷PBS中。在一个末端（颈部末端）快速抓住子宫肌层，并在肌肉层和蜕膜组织之间滑动精细剪刀，以释放与周围蜕膜组织的胚胎。 注意:肌肉倾向于快速收缩提取，所以这一步必须尽可能快。 </li><li>使用一对细镊子切断Reichert膜和胎盘。 </li><li> 轻轻取出卵黄囊，并将其置于含有0.5mL消化混合物的24孔组织培养板中。 注意:在这一步，可以收集胚胎血液。 <ol><li>在切断脐带和卵黄血管后立即将胚胎转移到含有10mM冰冷的乙二胺四乙酸（EDTA）的12孔组织培养板中。使用锋利的精细剪刀斩断胚胎，尽量限制组织扩张。在冰上孵育10-15分钟，收集含有血液的EDTA。 </li></ol></li><li>取出胚胎周围的羊膜</li><li>对于阶段&lt;E11.5，计算体细胞对数以更好地分期胚胎和更好的时间分辨率（每个体细胞对在〜1小时30分钟内发展）。 </li><li>切胚胎我们的头镊子或精剪刀。将头部转移到具有0.5mL消化混合物的24孔板中。 注意:进一步解剖晚期阶段的神经外胚层和脑，用于流式细胞术分析;小心清除周围血管丛。 </li><li>切割后肢上方的胚胎并移除前肢。 </li><li> 要隔离肝脏，用一对精细的镊子打开胸部。夹住心脏前方，轻轻拉动，同时使用第二镊子将器官从身体释放出来。 <ol><li>仔细分离胎儿肝脏与心脏和肠道。将胎肝转移到具有0.5mL消化混合物的24孔板中。仔细监测解剖显微镜下的转移。 </li></ol></li><li>通过聚合酶链反应测定（PCR）收集尾区（或任何其他胚胎部分）进行基因分型。 注意:可以从E10.5胚胎收获其他组织和器官，以使用流式细胞术进行类似的分析。血，头滑雪n，主动脉 - 性腺 - 中肾区（AGM），心脏和神经外胚层可以在E10.5收集。在晚期阶段，也可以收集肺，肾，脾和胰腺。以前描述了不同发展阶段AGM解剖的详细描述14 。 </li></ol> 3.流式细胞术的胚胎组织加工<ol><li>在37℃下将器官（置于消化混合物中）孵育30分钟。 </li><li>将组织和酶溶液转移到置于装有6ml FACS缓冲液（0.5％牛血清白蛋白（BSA））和2mM EDTA的1×PBS中的6孔组织培养板中的100μm筛管上。通过用2mL注射器的黑色橡胶活塞轻轻地糖化来机械解离以获得单细胞悬浮液。 注意:从现在开始，所有步骤应在4°C进行。 </li><li>用巴斯德移液管收集细胞悬浮液，并转移到15 mL管中。 </li><li> SPI在4℃下，320℃，n进行7分钟。通过抽吸弃去上清液。 </li><li> 将沉淀物重悬于60μLFc阻断缓冲液（CD16 / CD32阻断抗体，在FACS缓冲液中稀释1/50）。 <ol><li>将50μL单孔细胞悬浮液每孔转移到圆底96多孔板中。在冰上孵育至少15分钟。 注意:处理少量样品时，也可以在5 mL聚苯乙烯FACS管中进行染色。 </li><li>将剩余的10μL转移到5 mL聚苯乙烯FACS管中，以获得每个组织的样品池。该池将用作对照（ 即每个荧光染料的未染色样品和荧光减去一个（FMO）对照）。将每个荧光灯减去一个（FMO）对照（每个荧光染料一个）50μL的池转移到96多孔板。 注意:每个FMO对照包含来自抗体面板的所有荧光染料偶联抗体，除了该抗体正在测量。 FMO用于识别门边界，并控制多色面板中的光谱重叠。为了正确地解决不同组织之间的背景和自发荧光的变化，重要的是从每个组织的样品池中制备这些对照。 YFP表达的适当对照是来自Cre阴性胚胎的样品，通过PCR基因分型证实。 </li></ol></li></ol>表面抗原染色<ol><li> 在FACS缓冲液中制备抗体混合物（表2）。每个样品准备50μL抗体混合物。 <ol><li>使用以下荧光染料偶联的抗体:抗CD45.2（克隆104）;抗CD11b（克隆M1 / 70）;抗F4 / 80（克隆BM8）;抗AA4.1（克隆AA4.1）;抗体试剂盒（克隆2B8）;和抗Ter119（克隆Ter119）。 </li><li>在FACS缓冲液中为FMO对照制备六种抗体混合物。每个对照样品准备50μL抗体混合物。 没有TE:抗体混合物中的抗体稀释浓度比材料表中指定的最终浓度高两倍。对于具有6个荧光染料偶联抗体的小组，制备6个FMO对照。 表2提供了一个管的抗体混合物和FMO对照的组成。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td></td><td colspan="7">抗体积（μL）（终体积50μL） </td></tr><tr><td>抗体</td><td>克隆</td><td>抗体混合</td><td> FMO CD45.2 </td><td> FMO CD11b </td><td> FMO F4 / 80 </td><td> FMO AA4.1 </td><td> FMO套件</td><td> FMO Ter119 </td></tr><tr><td>抗CD45.2 </td><td> 104 </td><td> 1 </td><td> 0 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td>抗CD11b </td><td> M1 / 70 </td><td> 0.5 </td><td> 0.5 </td><td> 0 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td></tr><tr><td>抗F4 / 80 </td><td> BM8 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 0 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td>抗AA4.1 </td><td> AA4.1 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 0 </td><td> 1 </td><td> 1 </td></tr><tr><td>防套件</td><td> 2B8 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0 </td><td> 0.5 </td></tr><tr><td>抗TER119 </td><td> TER119 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0 </td></tr><tr><td colspan="2"> FACS缓冲区</td><td> 45.5 </td><td> 46.5 </td><td> 46 </td><td> 46.5 </td><td> 46.5 </td><td> 46 </td><td> 46 </td></tr></tbody></table>表2:用于染色和荧光减1（FMO）对照的抗体体积。最终体积为50μL所需抗体积（μL）。 <ol start="2"><li>向96孔多孔板中的样品中加入50μL抗体混合物（染色时的最终体积为100μL）。轻轻移液两次，并在冰上孵育30分钟。 </li><li>在4℃下以320×g旋转96孔多孔板7分钟，弃去上清液。重悬于200μLFACS缓冲液中。 </li><li>用150μLFACS缓冲液重复洗涤步骤（步骤4.3）两次。 </li><li>将样品和对照（FMO和未染色的池样品）通过70μm过滤器过滤到5 ml聚苯乙烯管中。存放在冰上直到获得。 </li></ol> 5.通过流式细胞术鉴定EMP和YS衍生的巨噬细胞注意:该协议使用4激光流式细胞计数器等式进行了优化使用405nm紫激光，488nm蓝色激光，562nm黄色激光和638nm红色激光。 <ol><li>按照制造商的说明准备每个偶联抗体的补珠。使用未染色的样品和补偿珠来优化激光强度。 </li><li>为了识别门边界，对每个抗体使用FMO（荧光减去一个）对照。 </li><li>为了排除死细胞，在获取前1分钟向管（含有200μL染色细胞悬液）中加入1μLDAPI（1mg / ml）。使用强DAPI信号和前向散射参数（FSC）来排除死细胞和碎屑。 注意:使用流式细胞仪的软件在采集过程中绘制门。补偿矩阵和分析可以在使用其他市售软件进行采样后进行。 </li><li>使用前向和侧向散射（FSC vs SSC）来执行活体细胞和双倍体歧视从总事件。 </li><li>在对数刻度轴上创建荧光点图，绘制子门，首先排除红细胞（Ter119 + ），然后识别祖细胞（Kit + ）和造血细胞（CD45 + Kit neg ）（参见图1 ）。 </li><li>创建荧光直方图以量化YS（ 图2 ），胎肝（ 图3 ）和脑（ 图4 ）中不同确定群体之间YFP的标记效率。 </li></ol>

<ol>
	<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=Three+pathways+to+mature+macrophages+in+the+early+mouse+yolk+sac.">Three pathways to mature macrophages in the early mouse yolk sac.</a> Blood. 106 (9), 3004-3011 (2005).</li><li>Palis, J., Robertson, S., Kennedy, M., Wall, C., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+erythroid+and+myeloid+progenitors+in+the+yolk+sac+and+embryo+proper+of+the+mouse.">Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.</a> Development. 126 (22), 5073-5084 (1999).</li><li>Chen, 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=Erythroid/myeloid+progenitors+and+hematopoietic+stem+cells+originate+from+distinct+populations+of+endothelial+cells.">Erythroid/myeloid progenitors and hematopoietic stem cells originate from distinct populations of endothelial cells.</a> Cell Stem Cell. 9 (6), 541-552 (2011).</li><li>Frame, J. M., Fegan, K. H., Conway, S. J., McGrath, K. E., 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=Definitive+Hematopoiesis+in+the+Yolk+Sac+Emerges+from+Wnt-Responsive+Hemogenic+Endothelium+Independently+of+Circulation+and+Arterial+Identity.">Definitive Hematopoiesis in the Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium Independently of Circulation and Arterial Identity.</a> Stem Cells. , (2015).</li><li>Kieusseian, A., Brunetdela de la Grange, P., Burlen-Defranoux, O., Godin, I., Cumano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+hematopoietic+stem+cells+undergo+maturation+in+the+fetal+liver.">Immature hematopoietic stem cells undergo maturation in the fetal liver.</a> Development. 139 (19), 3521-3530 (2012).</li><li>Gomez Perdiguero, ., 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=Tissue-resident+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors.">Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.</a> Nature. 518 (7540), 547-551 (2015).</li><li>Hoeffel, 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=C-Myb(+)+erythro-myeloid+progenitor-derived+fetal+monocytes+give+rise+to+adult+tissue-resident+macrophages.">C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.</a> Immunity. 42 (4), 665-678 (2015).</li><li>Kierdorf, K., Prinz, M., Geissmann, F., Gomez Perdiguero, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+function+of+tissue+resident+macrophages+in+mice.">Development and function of tissue resident macrophages in mice.</a> Semin Immunol. 27 (6), 369-378 (2015).</li><li>Houssaint, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+mouse+hepatic+primordium.+II.+Extrinsic+origin+of+the+haemopoietic+cell+line.">Differentiation of the mouse hepatic primordium. II. Extrinsic origin of the haemopoietic cell line.</a> Cell Differ. 10 (5), 243-252 (1981).</li><li>Cumano, A., Godin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+hematopoietic+system.">Ontogeny of the hematopoietic system.</a> Annu Rev Immunol. 25, 745-785 (2007).</li><li>McGrath, K. 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+Sources+of+Hematopoietic+Progenitors+Emerge+before+HSCs+and+Provide+Functional+Blood+Cells+in+the+Mammalian+Embryo.">Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo.</a> Cell Rep. 11 (12), 1892-1904 (2015).</li><li>Schulz, 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=A+lineage+of+myeloid+cells+independent+of+Myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of Myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>Chevalier, C., Nicolas, J. F., Petit, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+delivery+of+4-hydroxy-tamoxifen+for+clonal+and+polyclonal+labeling+of+cells+of+the+surface+ectoderm,+skin,+and+hair+follicle.">Preparation and delivery of 4-hydroxy-tamoxifen for clonal and polyclonal labeling of cells of the surface ectoderm, skin, and hair follicle.</a> Methods Mol Biol. 1195, 239-245 (2014).</li><li>Bertrand, J. Y., Giroux, S., Cumano, A., Godin, I. <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+development+during+mouse+embryogenesis.">Hematopoietic stem cell development during mouse embryogenesis.</a> Methods Mol Med. 105, 273-288 (2005).</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=Characterization+of+purified+intraembryonic+hematopoietic+stem+cells+as+a+tool+to+define+their+site+of+origin.">Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin.</a> Proc Natl Acad Sci U S A. 102 (1), 134-139 (2005).</li><li>Ginhoux, 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=Fate+mapping+analysis+reveals+that+adult+microglia+derive+from+primitive+macrophages.">Fate mapping analysis reveals that adult microglia derive from primitive macrophages.</a> Science. 330 (6005), 841-845 (2010).</li><li>Nakamura, E., Nguyen, M. T., Mackem, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+tamoxifen-regulated+Cre+activity+in+mice+using+a+cartilage-specific+CreER(T)+to+assay+temporal+activity+windows+along+the+proximodistal+limb+skeleton.">Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton.</a> Dev Dyn. 235 (9), 2603-2612 (2006).</li><li>Qian, B. Z., 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=CCL2+recruits+inflammatory+monocytes+to+facilitate+breast-tumour+metastasis.">CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis.</a> Nature. 475 (7355), 222-225 (2011).</li><li>Yona, 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=Fate+mapping+reveals+origins+and+dynamics+of+monocytes+and+tissue+macrophages+under+homeostasis.">Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.</a> Immunity. 38 (1), 79-91 (2013).</li><li>Sheng, J., Ruedl, C., Karjalainen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+Tissue-Resident+Macrophages+Except+Microglia+Are+Derived+from+Fetal+Hematopoietic+Stem+Cells.">Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells.</a> Immunity. 43 (2), 382-393 (2015).</li></ol>

作者没有什么可以披露的。

造血前体细胞的不同波浪在短时间内部分重叠，这使得每次发育造血波对免疫细胞的贡献的分析技术上非常具有挑战性。 他莫昔芬诱导型Cre系统提供了以时间可诱导的方式标记特定细胞并在胚胎或成年人中进行谱系分析的机会，而不需要离体或体外培养或移植。在他莫昔芬诱导的Cre菌株中，在细菌Cre重组酶和人雌激素受体（CRE-ER）的配体结合结构域的突变形式?...

造血祖细胞在发育过程中有几个相继但重叠的波浪，其骨髓后代保持成年。首先，单能的"原始"祖细胞出现在E7.5-E8.25之间鼠标卵黄囊1,2和产生胚胎的巨噬细胞而没有任何单核细胞的中间。来自原始祖细胞的巨噬细胞是否持续存在于成年大脑中，因为小神经胶质细胞仍然是主动调查的一个主题。第二，红血球前体（EMPs）出现在E8.5的卵黄囊中，进入血液并定殖胚胎。从环境管理计划卵黄囊hemogenic内皮在Runx1的依赖性的内皮到造血过渡3,4出现。虽然EMP可以分化成卵黄囊中的巨噬细胞，但它们也从胚胎期（E）9 5定殖于胎肝，并且不同分化成红细胞，巨核细胞，巨噬细胞，单核细胞粒细胞和肥大细胞6 。来自EMP的巨噬细胞在发育和成体组织中表现出增殖能力。是否EMP源性巨噬细胞分化绕过的单核细胞级仍有争议非常知之甚少其分化途径7,8。最后，造血干细胞（HSCs）在E10.5出现在主动脉 - 性腺 - 中肾区域内的胚胎中，并迁移到胎儿肝脏。长期再增殖能力的HSC仅检测到E11后（在42体节对阶段）9。在那里，他们扩大和区分E12.5直到明确的造血开始转移到骨髓，这在产后生活期间成为血细胞生产的主要部位10 。 sp妊娠和时间重叠的出现以及共同的免疫表型标志物迄今阻碍了我们区分这些胚胎造血祖细胞波的特异贡献的能力。虽然EMP和HSC都以Runx1依赖性方式产生，并表达转录因子Myb和生长因子受体Csf1r（集落刺激因子1受体，也称为巨噬细胞集落刺激因子受体）等，EMP可以区分为HSC由于缺乏淋巴细胞的潜力， 体外和体内都缺乏长期的重建潜力，缺乏表型谱系Sca- 11的表达 。需要遗传命运映射模型来表征巨噬细胞个体发育，因为它们允许以细胞特异性和时间特异性方式靶向胚胎祖细胞。我们在这里介绍我们实验室使用的命运映射协议来区分两个谱系在大多数成年组织中发现的巨噬细胞:HSC衍生的浸润巨噬细胞和不依赖HSC的宿主巨噬细胞。 组织定居巨噬细胞已被追溯到表达细胞因子受体12 CSF1R的Myb独立前体细胞和存在于使用三个互补命运-映射策略6在E8.5-E10.5胚胎。为了研究没有标记胎儿HSC的卵黄囊造血，我们使用转基因株式Cs r r r re re re er er of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of Csf1r启动子。因此，Cre重组酶在有限的时间窗口内将在Csf1r表达细胞中活跃。当与含有lox-STOP-lox盒（Rosa26 LSL-eYFP ）下游的荧光蛋白的报道菌株一起使用时，它将导致永久性在诱导时存在的细胞的遗传标记，也是其后代。给予E8.5活性形式的他莫昔芬，4-羟基他莫昔芬（OH-TAM），标记EMP和巨噬细胞，而不标记卵黄囊单能"原始"祖细胞或胎儿HSC。因此，我们已经描述了胚胎发育过程中EMPs及其后代的免疫表型，并评估了卵黄囊衍生的巨噬细胞对成年巨噬细胞池的贡献。需要进一步的工作来表征原始祖细胞来源的巨噬细胞是否也使用这种方法进行标记，以及它们是否可以促成成年巨噬细胞池。

<table><tbody><tr><td>#5 Straight Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>MC19/B Pascheff-Wolff Spring Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15371-92</td><td></td></tr><tr><td>8.5 cm straight scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>Anti-Mouse Kit-PE (clone 2B8)</td><td>BD Pharmingen</td><td>553355</td><td>1/200 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse CD45.2 APC-Cy7 (clone 104)</td><td>Sony</td><td>1149120</td><td>1/100 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse AA4.1-APC (CD93, clone AA4.1)</td><td>eBioscience</td><td>17-5892</td><td>1/100 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse Ter119 PerCP-Cy5.5 (clone Ter119)</td><td>Biolegend</td><td>560512</td><td>1/200 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse F4/80-BV421 (clone BM8)</td><td>BD Pharmingen</td><td>123137</td><td>1/100 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse CD11b PE-Cy7 (clone M1/70)</td><td>BD Pharmingen</td><td>552850</td><td>1/200 dilution in FACs Buffer</td></tr><tr><td>Anti-Mouse CD16/CD32 (Mouse BD Fc Block, clone 2.4G2)</td><td>BD Biosciences</td><td>553142</td><td></td></tr><tr><td>PBS 1x</td><td>Fischer Scientific</td><td>12559069</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fischer Scientific</td><td>11570506</td><td></td></tr><tr><td>Collagenase D&amp;nbsp;</td><td>Roche</td><td>11088882001</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Sigma</td><td>D4527-20KU</td><td></td></tr><tr><td>6-well tissue culture plate</td><td>Dutscher Dominique</td><td>353046</td><td></td></tr><tr><td>12-well tissue culture plate</td><td>Dutscher Dominique</td><td>353224</td><td></td></tr><tr><td>24-well tissue culture plate</td><td>Fischer Scientific</td><td>11874235</td><td></td></tr><tr><td>96 wells U-shape bottom tissue culture plate</td><td>Dutscher Dominique</td><td>353227</td><td></td></tr><tr><td>&amp;nbsp;Syringe Plastipak 2 mL&amp;nbsp;</td><td>Dutscher Dominique</td><td>300185</td><td></td></tr><tr><td>Nylon grid 100 &amp;micro;m cell strainers</td><td>Dutscher Dominique</td><td>352360</td><td></td></tr><tr><td>Nylon grid 70 &amp;micro;m cell strainers</td><td>Dutscher Dominique</td><td>352350</td><td></td></tr><tr><td>15 ml Falcon tubes</td><td>Dutscher Dominique</td><td>352096</td><td></td></tr><tr><td>Stericup GP Millipore filtration kit, 0.2 &amp;mu;m</td><td>Dutscher Dominique</td><td>51246</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906-500G</td><td></td></tr><tr><td>(Z)-4-HYDROXYTAMOXIFEN 25mg</td><td>Sigma</td><td>H7904-25MG</td><td>Special care should be taken when preparing and working with tamoxifen and its derivates. Always use appropriate personal protective equipment</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P3972</td><td>Always use appropriate personal protective equipment</td></tr><tr><td>PEG-35 castor oil (Kolliphor/Cremophor EL)</td><td>Sigma</td><td>C5135</td><td></td></tr><tr><td>Sunflower oil</td><td>Sigma</td><td>S5007-250ML</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma</td><td>24103-1L-R-D</td><td>Always use appropriate personal protective equipment and use under the fumehood</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E9884-500G</td><td></td></tr><tr><td>DAPI, 1 ML (1 MG/ML IN WATER)</td><td>Fischer Scientific</td><td>10116287</td><td></td></tr><tr><td>CytoFLEX S B2-R3-V4-Y4 flow cytometer</td><td>Beckman Coulter</td><td>B75408</td><td></td></tr><tr><td>CytoFLEX Daily QC Fluorospheres</td><td>Beckman Coulter&amp;nbsp;</td><td>B53230</td><td></td></tr><tr><td>VersaComp Antibody Capture Bead kit (2x5 mL)</td><td>Beckman Coulter&amp;nbsp;</td><td>B22804</td><td></td></tr><tr><td>FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J</td><td>The Jackson laboratory</td><td>19098</td><td></td></tr><tr><td>B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td><td>The Jackson laboratory</td><td>6148</td><td></td></tr></tbody></table>

identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry

巨噬细胞是免疫系统固有的专业吞噬细胞。在稳态状态下，无性巨噬细胞在成体组织中发现，其中它们作为感染和组织损伤的前线哨点。虽然其他免疫细胞从位于骨髓中的造血干细胞和祖细胞（HSPC）不断更新，但已经显示出巨噬细胞谱系，被称为驻留巨噬细胞，而不是从骨髓HSPC输入的组织中自我维持。这个谱系的例子是脑中的小胶质细胞，肝脏中的枯否细胞和表皮中的朗格汉斯细胞等。肠和结肠固有层是唯一没有HSPC独立的巨噬细胞的成体组织。最近的研究已经确定，存在的巨噬细胞来自不同于胎儿造血干细胞（HSC）的祖细胞的胚外卵黄囊造血。在卵黄囊定型造血中，红细胞生成素祖细胞（EMP）会引起红细胞和骨髓细胞，特别是驻极体巨噬细胞。 EMP仅在发育E8.5和E10.5天之间的蛋黄囊内产生，并且早在循环连接时迁移到胎肝，其中它们扩展和分化直到至少E16.5。他们的后代包括红细胞，巨噬细胞，嗜中性粒细胞和肥大细胞，但只有EMP来源的巨噬细胞持续到组织中成年。 EMP出现的瞬态性和与HSC生成的时间重叠使得这些祖细胞的分析困难。我们建立了基于巨噬细胞因子受体Csf1r启动子表达的他莫昔芬诱导的命运映射方案，通过流式细胞术在体内表征EMP和EMP来源的细胞。

通过将OH-TAM的E8.5施用于与携带Rosa26-LSL-eYFP报告物的雄性配对的Csf1r-Mer-iCre-Mer雌性中实现遗传命运测绘。在存在OH-TAM的情况下，切除盒可导致表达Csf1r的细胞中YFP的永久表达。我们从E10.5的胚胎收集了两个造血组织:卵黄囊和胎肝以及非造血组织（神经外胚层）。通过酶学和机械解离获得单细胞悬浮液，并用荧光偶联抗体染色后通过流式细胞术分析样品。排除死细胞和?...

Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

当浸润的巨噬细胞从循环前体连续招募到成体组织时，驻留的巨噬细胞在发育过程中种植其组织，在那里它们被维持而无需进一步从祖细胞输入。最近确定了居民巨噬细胞的祖细胞。在这里，我们提出了驻地巨噬细胞祖细胞的遗传命运映射方法。

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

identification-erythromyeloid-progenitors-their-progeny-mouse-embryo

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

12.9K 次观看.  Institut Pasteur. 当浸润的巨噬细胞从循环前体连续招募到成体组织时，驻留的巨噬细胞在发育过程中种植其组织，在那里它们被维持而无需进一步从祖细胞输入。最近确定了居民巨噬细胞的祖细胞。在这里，我们提出了驻地巨噬细胞祖细胞的遗传命运映射方法。 

视频: Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

步特定老鼠的精子细胞分选流式细胞仪

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

流式细胞术从小鼠组织中鉴定和分离爆破形成单元和集落形成单元红系祖细胞

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

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

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

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

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

生物学

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

免疫与感染

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

小鼠B细胞发育的流式细胞学表征

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

一个小鼠红细胞亚群富集去核活动由多光谱成像流式细胞仪鉴定

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

小鼠骨髓造血干细胞和祖细胞以及基质生态位细胞的流式细胞术分析

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...