Name: identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry
Uploaded: 2017-07-17T12:30:00
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?...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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>&#160;Syringe Plastipak 2 mL&#160;</td>
			<td>Dutscher Dominique</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon grid 100 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon grid 70 &#181;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 &#956;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&#160;</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&#160;</td>
			<td>B53230</td>
			<td></td>
		</tr>
		<tr>
			<td>VersaComp Antibody Capture Bead kit (2x5 mL)</td>
			<td>Beckman Coulter&#160;</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?...

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 ...

Research

JoVE Journal

Developmental Biology

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

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

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

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

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

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 ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

隔离并通过流式细胞仪分析胰腺间充质细胞

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...