Name: flow cytometry analysis of murine bone marrow hematopoietic stem and progenitor cells and stromal niche cells
Uploaded: 2022-09-28T12:50:00
Description: Watch this Scientific Journal Video about Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells at JoVE.com

LM得到了塔塔夫人纪念信托基金的资助。JR得到了科学与技术基金会（FCT;FCT 奖学金 UI/BD/150833/2021）。ML得到了FCT的博士奖学金（FCT奖学金 2021.04773.BD）的支持。DD得到了美国血液学会，Pablove基金会，FCT（EXPL/MED-ONC/0522/2021）和葡萄牙血液学会的资助。我们感谢i3s的TRACY设施的Catarina Meireles博士和Emilia Cardoso的支持。

该协议中使用的动物在12小时明暗循环和温度受控环境中，在特定无病原体条件下饲养在i3S动物设施中。免费提供标准的啮齿动物食物和水。所有动物都根据欧洲实验动物科学协会联合会概述的实验动物护理和处理标准（欧盟指令2010/63 / EU）接受了人道护理。对动物进行的实验程序（安乐死）得到了i3S动物伦理委员会（参考文献DD_2019_15）和Direção-Geral de Alimentação e Veterinária的批准。本协议中使?...

<ol>
	<li>Comazzetto, S., Shen, B., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niches+that+regulate+stem+cells+and+hematopoiesis+in+adult+bone+marrow.">Niches that regulate stem cells and hematopoiesis in adult bone marrow.</a> Developmental Cell. 56 (13), 1848-1860 (2021).</li><li>Purton, L. E., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+factors+in+murine+hematopoietic+stem+cell+assays.">Limiting factors in murine hematopoietic stem cell assays.</a> Cell Stem Cell. 1 (3), 263-270 (2007).</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 (7), 1109-1121 (2005).</li><li>Asada, 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=Differential+cytokine+contributions+of+perivascular+haematopoietic+stem+cell+niches.">Differential cytokine contributions of perivascular haematopoietic stem cell niches.</a> Nature Cell Biology. 19 (3), 214-223 (2017).</li><li>Mosteo, 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+dynamic+interface+between+the+bone+marrow+vascular+niche+and+hematopoietic+stem+cells+in+myeloid+malignancy.">The dynamic interface between the bone marrow vascular niche and hematopoietic stem cells in myeloid malignancy.</a> Frontiers in Cell and Developmental Biology. 9, 635189 (2021).</li><li>Christodoulou, 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=Live-animal+imaging+of+native+haematopoietic+stem+and+progenitor+cells.">Live-animal imaging of native haematopoietic stem and progenitor cells.</a> Nature. 578 (7794), 278-283 (2020).</li><li>Itkin, T., 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+bone+marrow+blood+vessels+differentially+regulate+haematopoiesis.">Distinct bone marrow blood vessels differentially regulate haematopoiesis.</a> Nature. 532 (7599), 323-328 (2016).</li><li>Kusumbe, A. P., 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=Age-dependent+modulation+of+vascular+niches+for+haematopoietic+stem+cells.">Age-dependent modulation of vascular niches for haematopoietic stem cells.</a> Nature. 532 (7599), 380-384 (2016).</li><li>Duarte, 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=Inhibition+of+endosteal+vascular+niche+remodeling+rescues+hematopoietic+stem+cell+loss+in+AML.">Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in AML.</a> Cell Stem Cell. 22 (1), 64-77 (2018).</li><li>Busch, K., 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=Fundamental+properties+of+unperturbed+haematopoiesis+from+stem+cells+in+vivo.">Fundamental properties of unperturbed haematopoiesis from stem cells in vivo.</a> Nature. 518 (7540), 542-546 (2015).</li><li>Laurenti, E., Gottgens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+haematopoietic+stem+cells+to+complex+differentiation+landscapes.">From haematopoietic stem cells to complex differentiation landscapes.</a> Nature. 553 (7689), 418-426 (2018).</li><li>Pietras, E. 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=Functionally+distinct+subsets+of+lineage-biased+multipotent+progenitors+control+blood+production+in+normal+and+regenerative+conditions.">Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.</a> Cell Stem Cell. 17 (1), 35-46 (2015).</li><li>Challen, G. A., Pietras, E. M., Wallscheid, N. C., Signer, R. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+murine+multipotent+progenitor+isolation+scheme:+Establishing+a+consensus+approach+for+multipotent+progenitor+identification.">Simplified murine multipotent progenitor isolation scheme: Establishing a consensus approach for multipotent progenitor identification.</a> Experimental Hematology. 104, 55-63 (2021).</li><li>Mayle, A., Luo, M., Jeong, M., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+murine+hematopoietic+stem+cells.">Flow cytometry analysis of murine hematopoietic stem cells.</a> Cytometry A. 83 (1), 27-37 (2013).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Hawkins, E. 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=Measuring+lymphocyte+proliferation,+survival+and+differentiation+using+CFSE+time-series+data.">Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.</a> Nature Protocols. 2 (9), 2057-2067 (2007).</li><li>Akinduro, 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=Proliferation+dynamics+of+acute+myeloid+leukaemia+and+haematopoietic+progenitors+competing+for+bone+marrow+space.">Proliferation dynamics of acute myeloid leukaemia and haematopoietic progenitors competing for bone marrow space.</a> Nature Communications. 9 (1), 519 (2018).</li><li>Beerman, 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=Functionally+distinct+hematopoietic+stem+cells+modulate+hematopoietic+lineage+potential+during+aging+by+a+mechanism+of+clonal+expansion.">Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (12), 5465-5470 (2010).</li><li>Gomariz, 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=Quantitative+spatial+analysis+of+haematopoiesis-regulating+stromal+cells+in+the+bone+marrow+microenvironment+by+3D+microscopy.">Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy.</a> Nature Communications. 9 (1), 2532 (2018).</li><li>Xu, 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=Stem+cell+factor+is+selectively+secreted+by+arterial+endothelial+cells+in+bone+marrow.">Stem cell factor is selectively secreted by arterial endothelial cells in bone marrow.</a> Nature Communications. 9 (1), 2449 (2018).</li><li>Amend, S. R., Valkenburg, K. C., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+hind+limb+long+bone+dissection+and+bone+marrow+isolation.">Murine hind limb long bone dissection and bone marrow isolation.</a> Journal of Visualized Experiments. (110), e53936 (2016).</li><li>Wang, 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=Ephrin-B2+controls+VEGF-induced+angiogenesis+and+lymphangiogenesis.">Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis.</a> Nature. 465 (7297), 483-486 (2010).</li><li>DeFalco, 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=Virus-assisted+mapping+of+neural+inputs+to+a+feeding+center+in+the+hypothalamus.">Virus-assisted mapping of neural inputs to a feeding center in the hypothalamus.</a> Science. 291 (5513), 2608-2613 (2001).</li><li>Boyer, S. W., Schroeder, A. V., Smith-Berdan, S., Forsberg, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+hematopoietic+cells+develop+from+hematopoietic+stem+cells+through+Flk2/Flt3-positive+progenitor+cells.">All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.</a> Cell Stem Cell. 9 (1), 64-73 (2011).</li><li>Siegemund, S., Shepherd, J., Xiao, C., Sauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hCD2-iCre+and+Vav-iCre+mediated+gene+recombination+patterns+in+murine+hematopoietic+cells.">hCD2-iCre and Vav-iCre mediated gene recombination patterns in murine hematopoietic cells.</a> PLoS One. 10 (4), 0124661 (2015).</li></ol>

虽然所描述的协议简单易行，但应特别注意具体步骤。例如，当获得冲洗的BM（步骤5.2）时，不应超过使PBS 2%FBS通过骨中央部分内部的指示体积或次数，因为这可能导致冲洗的样品被内膜细胞群严重污染。
可以对协议进行更改，以方便研究者执行协议。在样品提取（第2节）中，腿和/或骨骼可以在4°C下储存在PBS 2%FBS中长达24小时，然后再进行样品处理和分析。但是，应尽可能?...

流式细胞术是表征和前瞻性分离免疫细胞和造血细胞的宝贵方法。它也越来越多地用于分析不同组织的基质和上皮群体。造血干细胞（HSC）具有自我更新和多能性的独特特性。在成年哺乳动物中，HSC主要存在于骨髓（BM）中，在那里它们接收来自周围微环境或生态位的静止和生存信号1。HSC根据功能测定2正式定义。然而，一些具有里程碑意义的论文已经证明了流式细胞术在鉴定HSCs方面的有用性。通过使用有限的细胞表面标志物，可以区分HSC3中高度富集的造血群体。因此，流式细胞术是干细胞领域的中心方法。它已被广泛用于评估推定的生态位细胞类型和生态位因子对HSC的影响。通过将流式细胞术与成像和功能测定相结合，已经表明HSC得到了血管周围间充质干细胞（MSCs）和内皮细胞（ECs）的关键支持。BM MSCs是一个异质性群体，具有不同的细胞因子贡献4，但众所周知，瘦素受体（LepR）+ MSCs是关键的生态位细胞1。BM EC也是高度异质的，可以是正弦波，小动脉和H型/过渡血管的一部分5。不同的研究表明，这些不同的ECs的细微贡献。例如，内硬性正弦 EC 在空间上更接近静态 HSC6，而活性氧水平较低的非迁移性 HSC 位于小动脉EC 7 附近。壁龛的内膜与中心位置也非常重要。H型内膜血管与血管周围基质细胞有关，血管周围基质细胞随着年龄的增长而丢失，导致HSC丢失8。在急性髓系白血病中，中枢EC扩张，而内膜血管和内硬性HSC丢失9。
该领域的大多数研究都集中在造血本身和细胞对HSC的外在调节上。然而，人们越来越认识到，有必要更好地表征调节其他祖细胞（即多能祖细胞（MPP））的生态位，特别是考虑到它们是稳态造血的主要驱动因素10。与固定的分层结构相比，最近的研究表明，造血是一个连续体，其中HSC在早期分化为有偏差的MPP11。MPPs以不同的分类方案命名12，但国际实验血液学会（ISEH）最近的一篇共识论文提出，MPPs被区分为早期MPP，并根据其淋巴（MPP-Ly），巨核细胞和红系（MPP-Mk/E）和骨髓（MPP-G/M）偏差13。流式细胞术的使用对于进一步研究BM生态位在这些群体调节中的重要性至关重要。目前的流式细胞术方法使用可变门控策略来区分HSPC，并使用不一致的标记物鉴定基质细胞，即EC。当前方法的目标是提供一种简单且可重现的BM染色工作流程，以鉴定HSPC亚群，异质EC组和LepR + MSC。我们认为，尽管该技术与先前报道的方法（参见例如参考文献14）相当，但为两个功能性骨髓区域的造血细胞（内硬膜和中央BM8，15）以及BM基质生态位细胞的表型分析提供了一种更新且易于实施的方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 647 anti-mouse CD54/ICAM-1 antibody</td>
			<td>BioLegend</td>
			<td>116114</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Streptavidin</td>
			<td>BioLegend</td>
			<td>405207</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cyanine7 anti-mouse CD117 (c-kit) antibody</td>
			<td>BioLegend</td>
			<td>105826</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cyanine7 anti-mouse CD45 antibody</td>
			<td>BioLegend</td>
			<td>103116</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cyanine7 anti-mouse TER-119/erythroid cells antibody</td>
			<td>BioLegend</td>
			<td>116223</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse CD3&#949; antibody</td>
			<td>BioLegend</td>
			<td>100304</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse CD4 antibody</td>
			<td>BioLegend</td>
			<td>100404</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse CD8a antibody</td>
			<td>BioLegend</td>
			<td>100704</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse Ly-6G/Ly-6C (Gr-1) antibody</td>
			<td>BioLegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse TER-119/erythroid cells antibody</td>
			<td>BioLegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse/human CD11b antibody</td>
			<td>BioLegend</td>
			<td>101204</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse/human CD45R/B220 antibody</td>
			<td>BioLegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 510 anti-mouse CD150 (SLAM) antibody</td>
			<td>BioLegend</td>
			<td>115929</td>
			<td></td>
		</tr>
		<tr>
			<td>Calibrite 2 Color Beads</td>
			<td>BD Biosciences</td>
			<td>349502</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Merck Life Science</td>
			<td>C1889</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Merck Life Science</td>
			<td>D4693</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin</td>
			<td>ThermoFisher Scientific</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Leptin R Biotinylated Antibody</td>
			<td>R&#38;D systems</td>
			<td>BAF497</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue Fixed Cell Reagent (DAPI)</td>
			<td>ThermoFisher Scientific</td>
			<td>R37606</td>
			<td>DAPI reagent</td>
		</tr>
		<tr>
			<td>PE anti-mouse endomucin antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>12-5851-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse Flk2 (CD135)</td>
			<td>ThermoFisher Scientific</td>
			<td>12-1351-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine7 anti-mouse CD31 antibody</td>
			<td>BioLegend</td>
			<td>102524</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine7 anti-mouse CD48 antibody</td>
			<td>BioLegend</td>
			<td>103424</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP anti-mouse Ly-6A/E (Sca-1) antibody</td>
			<td>BioLegend</td>
			<td>108122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) tablets</td>
			<td>Merck Life Science</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse CD16/32 antibody</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer 10x</td>
			<td>BioLegend</td>
			<td>420302</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Violet Fixable Viability Dye</td>
			<td>BioLegend</td>
			<td>423114</td>
			<td>fluorescent dye</td>
		</tr>
	</tbody>
</table>

flow cytometry analysis of murine bone marrow hematopoietic stem and progenitor cells and stromal niche cells

骨髓（BM）是在骨骼中发现的软组织，造血是产生新血细胞的过程，主要发生于此。因此，它含有造血干细胞和祖细胞（HSPC），以及有助于维持和调节HSPC的支持基质细胞。 血液学和其他BM疾病通过直接影响造血细胞和/或通过改变BM生态位来破坏造血。在这里，我们描述了一种通过流式细胞术对小鼠BM HSPC和基质生态位群体进行表型分析来研究健康和恶性肿瘤造血的方法。我们的方法详细介绍了在内膜和中枢BM组分中富集BM细胞所需的步骤，以及适当的门控策略，以确定参与HSPC调节的两种关键生态位细胞类型，内皮细胞和间充质干细胞。这里提出的表型分析可以与小鼠突变体，疾病模型和功能测定相结合，以表征HSPC区室及其生态位。

健康年轻成年C57Bl/6小鼠HSC和MPP的流式细胞术分析代表性图如图 1所示。门控策略遵循ISEH13提出的最新协调命名法。在分析干扰（例如感染或癌症）的影响时，使用对照小鼠作为正常门的参考非常重要。荧光负一（FMO）控制对于描绘门的边界特别有用，但应该注意的是，盲目地基于FMO设置门限可能会省略靶细胞或包括非靶细胞。例如，CD48集的强度越低，静态...

Watch this Scientific Journal Video about Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells at JoVE.com

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

在这里，我们描述了一种简单的方案，用于将小鼠骨髓细胞分离和染色为表型造血干细胞和祖细胞以及支持生态位内皮和间充质干细胞。还包括富集位于内膜和中央骨髓区域的细胞的方法。

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 ...

所述协议允许对骨髓造血干和祖细胞以及骨髓基质细胞进行表型分析。它可用于研究不同环境中对这些人群的扰动。与先前描述的其他方法相比，该技术允许对造血细胞进行表型分析，特别是在两个功能性骨髓区域，内膜和中央骨髓以及骨髓基质细胞。首先，将安乐死的动物腹部朝上放在培养皿上，并喷洒70%乙醇。用剪刀在腹部上方切开，将皮肤一直拉到脚踝。抓住其中一条腿，用剪刀剪开它的底部，将其与动物分开。然后通过切割脚踝将脚从腿上移开，并将腿放在冰冷的PBS 2%FBS中。接下来，用镊子抓住髋骨并在它后面剪开，用剪刀将其与动物分开。将髋骨放入冰冷的PBS 2%FBS中。将腿骨和髋骨放入培养皿中后，使用无菌手术刀清洁骨头。然后用手术刀在膝盖上切开胫骨和股骨，并将干净的骨头放入冰冷的PBS 2%FBS中。将股骨或胫骨放入装有冰冷PBS 2%FBS的研钵中，并用研杵将其轻轻按压在研钵壁上，将骨头压碎。接下来，使用10毫升移液器上下移液所得细胞悬液以均质化并通过放置在管顶部的40微米细胞过滤器将其转移到50毫升管中。用更多的PBS 2%FBS冲洗砂浆，并通过相同的40微米细胞过滤器将溶液转移到相同的50毫升管中。将 50 毫升管以 500 G 在 4 摄氏度下离心五分钟。弃去上清液后，在室温下上下移液数次，将细胞沉淀重悬于5毫升红细胞裂解缓冲液中。在室温下孵育两分钟后，加入15毫升PBS 2%FBS并离心50毫升管。如果细胞沉淀看起来不发红，则弃去上清液，将沉淀重悬于200微升PBS 2%FBS中，并将细胞悬液转移到96孔V底板的孔中进行染色。如前所述压碎骨头后，用剪刀剪下P1000移液器的尖端，并用它来将研钵的所有内容物转移到50毫升的管中。然后将 50 毫升管以 500 G 在 4 摄氏度下离心五分钟。弃去上清液后，将沉淀重悬于三毫升胶原酶IV和该分散酶II溶液中。将试管在 37 摄氏度下孵育 40 分钟。用PBS 2%FBS将试管加满至25毫升，然后涡旋混合。接下来，通过100微米的细胞过滤器将内容物转移到新的50毫升管中。然后将15毫升PBS 2%FBS加入旧的50毫升管中，并通过100微米细胞过滤器将溶液转移到新的50毫升管中。离心并弃去上清液后，用移液管上下移液数次，将细胞沉淀重悬于5毫升红细胞裂解缓冲液中。在室温下孵育两分钟后，加入15毫升PBS 2%FBS。再次，离心管，如果丢弃上清液后细胞沉淀看起来不发红，则将沉淀重悬于200微升PBS 2%FBS中，并将细胞悬液转移到96孔V底板的孔中进行染色。然后将板以500 G在4摄氏度下离心三分钟。将股骨放在培养皿上，并使用无菌手术刀切割骨头的末端。然后使用无死体积0.5毫升胰岛素注射器将100微升PBS 2%FBS从每侧穿过骨内部一次，并将溶液收集在含有1毫升PBS 2%FBS的微量离心管中，从而冲洗骨骼的中心部分。随后将细胞悬液转移到标记为含有4毫升PBS 2%FBS的冲洗骨髓的50毫升管中。用冰冷的PBS 2%FBS将骨头的末端用冰冷的PBS 2%FBS轻轻压在研钵壁上，将其压碎。细胞悬液均质化后，通过40微米细胞过滤器将其转移到标记为碎骨髓的50毫升管中。最后，用更多的PBS 2%FBS冲洗砂浆，并将溶液转移到同一管中。在健康的年轻成年C57BL6J小鼠的总骨髓中进行造血干细胞和多能祖细胞的流式细胞术分析。在分析总骨髓中的基质细胞、内皮细胞和间充质干细胞时，可以根据瘦素受体表达轻松鉴定间充质干细胞，根据CD31和SCA-1的表达可以鉴定内皮细胞。全骨髓内皮细胞的流式细胞术分析显示基于ICAM1的小动脉和正弦内皮细胞之间的区别。对中枢和内膜骨髓组织中动脉和正窦骨髓内皮细胞的定量表明，动脉骨髓内皮细胞在内分泌区域更丰富，而中央骨髓区域的正弦更常见。当获得冲洗的骨髓时，不应超过通过PBS 2%FBS通过骨中央部分内部的体积或次数。将所描述的方法与所分析的细胞群的功能测定相结合，将提供有关在研究条件下发生的这些群体的潜在扰动的进一步有价值的信息。所描述的方案允许对内膜和中央骨髓中的造血细胞进行差异性分析，使研究人员能够研究在这些骨髓位置可能发生的血位扰动。

Research

JoVE Journal

Biology

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

小鼠卵黄囊和AGM早期造血干细胞的分离

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Homing of Hematopoietic Cells to the Bone Marrow

归巢的骨髓造血细胞

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...