Name: isolation of mouse megakaryocyte progenitors
Uploaded: 2021-05-20T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Mouse Megakaryocyte Progenitors at JoVE.com

作者希望感谢Monique Freund，Catherine Ziessel和Ketty的技术援助。这项工作得到了ARMESA（医学和公共发展协会）和Grant ANR-17-CE14-0001-01的支持，以 Henri.de la。大厅。

涉及动物的协议是根据斯特拉斯堡大学动物实验伦理学CREMEAS委员会（斯特拉斯堡动物研究区教育委员会）进行的。许可证编号：E67-482-10）。
1. 小鼠骨骼采集
<ol><li>按照机构准则牺牲动物。 注：本手稿中提供的数据来自8至12周龄的C57Bl / 6小鼠。获得的细胞数量和被引群体的频率可能随年龄和小鼠品系而变化。</li>
	<li>用70%乙醇喷洒身体。</li>
	<li>使用剪刀，在垂直于脊柱?...

<ol>
	<li>Kaushansky, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+review:+megakaryopoiesis+and+thrombopoiesis.">Historical review: megakaryopoiesis and thrombopoiesis.</a> Blood. 111 (3), 981-986 (2008).</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 (6774), 193-197 (2000).</li><li>Debili, 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=Characterization+of+a+bipotent+erythro-megakaryocytic+progenitor+in+human+bone+marrow.">Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow.</a> Blood. 88 (4), 1284-1296 (1996).</li><li>Forsberg, E. C., Serwold, T., Kogan, S., Weissman, I. L., Passegu&#233;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+evidence+supporting+megakaryocyte-erythrocyte+potential+of+flk2/flt3++multipotent+hematopoietic+progenitors.">New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors.</a> Cell. 126 (2), 415-426 (2006).</li><li>Vannucchi, A. 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=Identification+and+characterization+of+a+bipotent+(erythroid+and+megakaryocytic)+cell+precursor+from+the+spleen+of+phenylhydrazine-treated+mice.">Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.</a> Blood. 95 (8), 2559-2568 (2000).</li><li>Pronk, C. 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=Elucidation+of+the+phenotypic,+functional,+and+molecular+topography+of+a+myeloerythroid+progenitor+cell+hierarchy.">Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.</a> Cell Stem Cell. 1 (4), 428-442 (2007).</li><li>Nakorn, T. N., 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=Characterization+of+mouse+clonogenic+megakaryocyte+progenitors.">Characterization of mouse clonogenic megakaryocyte progenitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (1), 205-210 (2003).</li><li>Ng, 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=Characterization+of+thrombopoietin+(TPO)-responsive+progenitor+cells+in+adult+mouse+bone+marrow+with+in+vivo+megakaryocyte+and+erythroid+potential.">Characterization of thrombopoietin (TPO)-responsive progenitor cells in adult mouse bone marrow with in vivo megakaryocyte and erythroid potential.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (7), 2364-2369 (2012).</li><li>Strassel, 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=Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors.">Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.</a> Experimental Cell Research. 318 (1), 25-32 (2012).</li><li>Brouard, 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=A+unique+microenvironment+in+the+developing+liver+supports+the+expansion+of+megakaryocyte+progenitors.">A unique microenvironment in the developing liver supports the expansion of megakaryocyte progenitors.</a> Blood Advances. 1 (21), 1854-1866 (2017).</li><li>Boscher, J., Gachet, C., Lanza, F., L&#233;on, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megakaryocyte+culture+in+3D+methylcellulose-based+hydrogel+to+improve+cell+maturation+and+study+the+impact+of+stiffness+and+confinement.">Megakaryocyte culture in 3D methylcellulose-based hydrogel to improve cell maturation and study the impact of stiffness and confinement.</a> Journal of Visualized Experiments:JOVE. , (2021).</li><li>Sanjuan-Pla, 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=Platelet-biased+stem+cells+reside+at+the+apex+of+the+haematopoietic+stem-cell+hierarchy.">Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.</a> Nature. 502 (7470), 232-236 (2013).</li><li>Haas, 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=Inflammation-driven+fast-track+differentiation+of+HSCs+into+the+megakaryocytic+lineage.">Inflammation-driven fast-track differentiation of HSCs into the megakaryocytic lineage.</a> Experimental Hematology. 42 (8), 14 (2014).</li><li>Shin, J. 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本文中描述的方法允许提取和纯化小鼠MEP和MKp。方案优化的一个重要参数是获得足够数量的细胞，这些细胞与大多数基于分子和细胞的测定相容。收集小鼠骨以进行造血细胞提取的一般做法通常包括收获每只小鼠的股骨和胫骨。因此，骨盆骨是造血材料的另一个来源，经常被忽视。不收集髂嵴的原因是对小鼠骨骼的内部解剖结构知之甚少，并且用户通常通过穿过或刚好在股骨头上方来收集后肢。?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62498">Isolation of Mouse Megakaryocyte Progenitors</a>. A figure was updated.
Figure 2 was updated from:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62498/62498fig02.jpg" /> 
Figure 2: Magnetic depletion of lineage committed (Lin) cells. (A) Schematic representation of the magnetic depletion protocol. First, unsorted bone marrow cells are labeled with the biotin-conjugated rat anti-mouse antibody cocktail. Cells are then incubated with anti-rat Ig coated magnetic beads and subsequently subjected to the magnetic depletion using a strong magnet. The magnet will retain the labeled magnetic Lin+ fraction against the tube walls, while the unlabeled non-magnetic Lin- negative fraction will be collected in a new tube. (B) Lineage committed cells can be identified using fluorescent conjugated streptavidin. Typical analysis of the lineage expression in cells prior to magnetic depletion (total bone marrow) and after magnetic depletion (Lin- Fraction) N = 21. <a href="https://www.jove.com/files/ftp_upload/62498/62498fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62498/62498fig2v2.jpg" /> 
Figure 2: Magnetic depletion of lineage committed (Lin) cells. (A) Schematic representation of the magnetic depletion protocol. First, unsorted bone marrow cells are labeled with the biotin-conjugated rat anti-mouse antibody cocktail. Cells are then incubated with anti-rat Ig coated magnetic beads and subsequently subjected to the magnetic depletion using a strong magnet. The magnet will retain the labeled magnetic Lin+ fraction against the tube walls, while the unlabeled non-magnetic Lin- negative fraction will be collected in a new tube. (B) Lineage committed cells can be identified using fluorescent conjugated streptavidin. Typical analysis of the lineage expression in cells prior to magnetic depletion (total bone marrow) and after magnetic depletion (Lin- Fraction) N = 21. <a href="https://www.jove.com/files/ftp_upload/62498/62498fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62498">Isolation of Mouse Megakaryocyte Progenitors</a>. A figure was updated.

Erratum: Isolation of Mouse Megakaryocyte Progenitors

血小板由巨核细胞产生。这些大的多倍体细胞位于骨髓中，对于所有血细胞，它们都来自造血干细胞（HSC）1。骨髓中巨核细胞产生的经典途径起源于HSC，涉及不同祖细胞的产生，这些祖细胞逐渐限制了它们的分化潜力2。签署对巨核细胞谱系承诺的第一个祖是巨核细胞 - 红细胞祖细胞（MEP），这是一种能够同时产生红细胞和巨核细胞3，4，5的双能祖。然后，MEP产生一个单能祖细胞/前体（MKp），它将分化成能够产生血小板的成熟巨核细胞。这些祖细胞的产生所涉及的机制，以及它们分化和成熟为巨核细胞是复杂的，只能部分理解。此外，MEP群体在分化潜力方面的异质性和这些细胞的内在承诺水平尚不清楚。为了破译这些过程，必须获得（或能够获得）纯化的MEP和MKp群体，以进行精细分子和单细胞分析。
一些研究表明，细胞表面标记物的特殊组合用于鉴定小鼠6，7，8中致力于巨核细胞谱系的祖细胞。从这些中设计出一种方法，允许从小鼠中纯化MEP和MKp。该方法经过优化，可获得足够数量和质量的细胞，以进行大量测定。考虑到伦理因素，为了尽量减少参与实验的动物数量，我们从股骨和胫骨以及髂嵴中收获骨髓。这种骨含有高频率和数量的造血祖细胞，并且在长骨收获过程中大部分时间都受损。这里介绍的是可靠收集该骨骼的详细方法。
优化的第二个标准是产生高度纯化的细胞群。荧光活化细胞分选（FACS）是获得纯化目标细胞群的首选方法。然而，当感兴趣的细胞群非常罕见时，就会达到低产量。因此，浓缩程序是必要的。在该协议中，使用磁珠选择负选择程序。

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			<td>21-gauge needles</td>
			<td>BD Microlance</td>
			<td>301155</td>
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			<td>7AAD</td>
			<td>Sigma-Aldrich</td>
			<td>A9400</td>
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			<td>eBioscience</td>
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			<td>Antibody B220-biotin</td>
			<td>eBioscience</td>
			<td>13-0452-85</td>
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			<td>eBioscience</td>
			<td>13-0112-85</td>
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			<td>eBioscience</td>
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			<td>eBioscience</td>
			<td>13-9766-82</td>
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			<td>eBioscience</td>
			<td>13-0081-85</td>
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			<td>eBioscience</td>
			<td>13-5921-85</td>
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			<td>eBioscience</td>
			<td>13-1271-85</td>
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			<td>eBioscience</td>
			<td>11-0451-85</td>
			<td>Cell sorting</td>
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			<td>eBioscience</td>
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			<td>Antibody TER119-APC</td>
			<td>eBioscience</td>
			<td>17-5921-83</td>
			<td>Cell sorting</td>
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			<td>Antibody CD45-PECy7</td>
			<td>eBioscience</td>
			<td>25-0451-82</td>
			<td>Cell sorting</td>
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			<td>Antibody CD45-biotin</td>
			<td>eBioscience</td>
			<td>13-0451-85</td>
			<td>Cell sorting</td>
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			<td>Antibody CD9-FITC</td>
			<td>eBioscience</td>
			<td>11-0091-82</td>
			<td>Cell sorting</td>
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			<td>Antibody&#160; c-kit-APC</td>
			<td>eBioscience</td>
			<td>17-1171-83</td>
			<td>Cell sorting</td>
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			<td>Antibody Sca-1-PE</td>
			<td>eBioscience</td>
			<td>12-5981-83</td>
			<td>Cell sorting</td>
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			<td>Antibody CD16/32-PE</td>
			<td>eBioscience</td>
			<td>12-0161-83</td>
			<td>Cell sorting</td>
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			<td>Antibody CD150-PECy7</td>
			<td>eBioscience</td>
			<td>25-1502-82</td>
			<td>Cell sorting</td>
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			<td>Culture medium StemSpan-SFEM</td>
			<td>Stemcell technologies</td>
			<td>#09650</td>
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			<td>Dissection pad</td>
			<td>Fisher Scientific</td>
			<td>10452395</td>
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			<td>Life Technologies</td>
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			<td>vWR Chemicals</td>
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			<td>Glass pasteur pipette</td>
			<td>Dutscher</td>
			<td>42011</td>
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			<td>Magnet :&#160; DynaMag-5</td>
			<td>Thermo Fisher Scientific</td>
			<td>12303D</td>
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			<td>Magnetic beads: Dynabeads Sheep Anti-Rat IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>11035</td>
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			<td>Megacult</td>
			<td>Stemcell technologies</td>
			<td>#04970</td>
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			<td>MethoCult SF M3436</td>
			<td>Stemcell technologies</td>
			<td>#03436</td>
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			<td>Newborn Calf Serum</td>
			<td>Dutscher</td>
			<td>50750-500</td>
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			<td>BD Bioscience</td>
			<td>555899</td>
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			<td>Fisher Scientific</td>
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			<td>Falcon</td>
			<td>352063</td>
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			<td>Sterile 5mL polystyrene tubes</td>
			<td>Falcon</td>
			<td>352054</td>
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			<td>Sterile tubes with 70&#181;m cell strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
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			<td>Sterile petri dish</td>
			<td>Falcon</td>
			<td>353003</td>
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			<td>554063</td>
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			<td>Tube roller</td>
			<td>Benchmark Scientific</td>
			<td>R3005</td>
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isolation of mouse megakaryocyte progenitors

骨髓巨核细胞是确保血小板产生的大多倍体细胞。它们通过巨核生成从造血干细胞中产生。该过程的最后阶段很复杂，通常涉及双能巨核细胞 - 红细胞祖细胞（MEP）和单能巨核细胞祖细胞（MKp）。这些群体先于 真正的 巨核细胞的形成，因此，它们的分离和表征可以允许对巨核细胞的形成进行稳健和公正的分析。该协议详细介绍了从小鼠骨髓收集造血细胞的程序，通过磁消耗富集造血祖细胞，最后是产生高度纯化的MEP和MKp群体的细胞分选策略。首先，骨髓细胞从股骨，胫骨和髂嵴收集，髂嵴是含有大量造血祖细胞的骨骼。髂骨的使用大大增加了每只小鼠获得的总细胞数，从而有助于更合乎道德地使用动物。使用450 nm磁珠优化了磁谱系耗尽，允许通过流式细胞术进行非常有效的细胞分选。最后，该协议提出了两种高度纯化的巨核细胞祖细胞群体分类的标记和门控策略：MEP（Lin-Sca-1-c-Kit+CD16 / 32-CD150+CD9dim）和MKp（Lin- Sca-1-c-Kit+CD16 / 32-CD150+CD9明亮）).该技术易于实施，并提供足够的细胞材料来执行i）分子表征以更深入地了解其身份和生物学，ii） 体外 分化测定，这将更好地理解巨核细胞成熟的机制，或iii）与微环境相互作用的 体外 模型。

通过流式细胞术对鉴定为MEP和MKp的细胞进行表型分析。细胞用CD41a和CD42c的荧光偶联抗体标记，CD41a和CD42c是巨核细胞和血小板谱系的经典标志物。这两种标记物均由MKp群体的细胞表达，而这些标记尚未在MEP群体的细胞表面检测到（图4Ai，4Aii）。多倍体是巨核细胞的标志。还分析了分类群体的DNA含量，并证明MEP群体的细胞大多为2N，MKp细胞的一小部分是4N，但在这?...

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Isolation of Mouse Megakaryocyte Progenitors

该方法描述了通过流式细胞术从小鼠股骨，胫骨和盆骨中纯化MEP和MKp。

小鼠巨核细胞祖细胞的分离

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...

分离关键的巨核细胞祖细胞群，可以在分子测定中对细胞培养物中的谱系层次结构进行精细分析，直至单细胞水平。该协议通过最大限度地减少所需的动物数量，在MEP和MKP群体的细胞分选的有效过程中结合了更合乎道德的做法。对于骨骼，收集喷洒8至12周龄C57黑六只小鼠的身体，含70%乙醇。使用剪刀在皮肤上做一个五到一厘米的切口，垂直于脊柱。然后撕扯整个身体周围的皮肤，把它拉下来。将鼠标面朝下放在解剖垫上，然后通过滑动手指或剪刀沿着暴露的脊柱从上到下定位骨盆骨。要定位髂嵴，请识别后肢附近腰部区域的小肿块。将剪刀平行于脊柱，贴在椎骨上，并靠近髂嵴凸起后，继续沿着骨盆骨上方的脊柱一侧切割肌肉，方法是将剪刀沿着椎骨向下滑动到尾巴。然后在椎骨和髂嵴之间切开，尽可能靠近椎骨。并切除剩余的肌肉，使肢体与身体分离。将分离的肢体转移到干净的表面上。按照机构指南丢弃身体的其余部分后，使用镊子和手术刀通过去除周围组织来暴露骨盆，股骨和胫骨。使用镊子固定股骨远端，并用手术刀轻轻切开关节周围的肌肉，小心地将股骨头与骨盆骨脱臼。刮掉骨盆骨的剩余肌肉，并在支撑股骨头的腔的中间切开。保留髂骨并丢弃骨骼的三角形薄侧。使用手术刀去除髂骨周围的残留组织，并将清洁的骨头放入补充有2%新生犊牛血清的无菌PBS中。然后用剪刀将脚从脚踝处的腿上切下来。用镊子握住胫骨的下部，将肌肉刮向膝盖。丢弃腓骨。然后用手术刀切开胫骨平台，并将胫骨置于无菌PBS 2%NBCS中。去除股骨周围的残余组织后，用镊子固定股骨的上侧，并将手术刀刀片放在膝盖骨的底部。向膝盖骨施加与股骨平行的力以分离膝盖骨。然后将股骨置于无菌PBS 2%NBCS中。在层流柜中，将骨头转移到充满无菌PBS 2%NBCS的无菌培养皿中。用手术刀切掉股骨头。用无菌PBS 2%NBCS填充一毫升注射器，并将21号针头连接到出口。然后用两毫升无菌PBS 2%NBCS填充五毫升聚丙烯管。用镊子握住股骨，将针头插入左侧凹槽中，通过旋转去除膝盖骨后，确保针头完全插入骨骼，直至斜角。针头插入后，将用针头将骨头转移到含有两毫升PBS 2%NBCS的管中。然后从注射器分配并抽吸PBS 2%NBCS，直到骨头清晰。从股骨上取下针头，然后将其插入股骨头所在的另一侧的孔中。分配并吸出缓冲液后，丢弃骨头。对于髂嵴和胫骨，使用镊子固定骨头，并通过旋转轻轻地将针头插入开口侧。确保针头完全插入骨头，直到斜角。然后将用针头将骨头转移到含有两毫升PBS 2%NBCS的管中。从注射器中分配并抽吸PBS 2%NBCS，直到骨骼清晰。将所有细胞悬浮液通过40微米的细胞过滤器盖，置于无菌的5毫升聚苯乙烯管上，并加入500微升PBS 2%NBCS。留出100微升细胞悬浮液作为总骨髓。然后将其储存在冰上以进行染色过程。通过离心沉淀过滤后的悬浮液。弃去上清液后，将沉淀重悬于新鲜制备的一抗混合物中，在冰上孵育30至45分钟。留出10微升细胞悬浮液放入无菌的5毫升聚苯乙烯管中，标记为Lin Pores Fraction，并加入90微升PBS 2%NBCS。同时，准备一颗用于磁耗竭的珠子，将它们重新悬浮在小瓶中，彻底涡旋30秒。将对应于每个靶细胞两个磁珠的体积转移到五毫升聚丙烯管中，并通过将管放在磁铁上，用PBS 2%NBCS洗涤磁珠两次。用无菌玻璃巴氏移液管除去洗涤缓冲液后，将微珠重悬于500微升无菌PBS 2%NBCS中。对于磁性耗尽的第一步，将250微升珠子加入洗涤细胞的沉淀中，并在冰上轻轻混合五分钟。然后加入两毫升无菌PBS 2%NBCS，轻轻混合，将管放入磁铁中两分钟。继续使用无菌玻璃巴氏移液器收集非磁性部分。对于磁耗竭的第二步，将其添加到剩余的250微升磁珠中。将用石蜡密封的管子放在管辊上，在四摄氏度下放置20分钟。然后加入两毫升无菌PBS 2%NBCS，轻轻混合。并将管子与磁铁放在一起两分钟。将非磁性馏分收集到无菌的五毫升聚丙烯管中，标记，无磁性的Lin Neg级分，用无菌玻璃巴氏移液管，然后继续进行细胞分选巨核细胞祖细胞，如文本手稿中所述。对于用于细胞分选的门控策略，在Lin Neg群体中，选择表达C-试剂盒的细胞以及Sca-1或CD16 / 32抗原的低表达或无表达。在该群体中，MEP被鉴定为CD150阳性和CD9昏暗细胞。MKP为CD150阳性，且CD9为亮细胞。在具有代表性的流式细胞术分析中，鉴定为MEP和Mkp的细胞被标记为巨核细胞和血小板谱系的CD41a和CD42c经典标记物的荧光偶联抗体。这两种标记物均由MKp群体的细胞表达，并且在MEP群体的细胞表面未被检测到。分类的巨核细胞的DNA含量表明，MEP群体的细胞大多为2n。MKp细胞的一小部分是外来的。在这些群体中未显着检测到较高的倍体细胞。在半固体克隆检测中，在MEP和MKp人群中都检测到CFU-MK。BFU-E在MKp群体中未检测到，但在MEP和CD150阴性CD9昏暗的祖细胞群中检测到。分化第三天的显微观察表明，MEP和MKp主要产生巨核细胞，被鉴定为大细胞。培养三天后获得的巨核细胞使用CD41和CD42c表达进行鉴定，分别代表MEP和MKp细胞群产生的54%和82%的细胞。与MEP群体相比，从MKp群体衍生的巨核细胞中产生的巨核细胞的倍数更大。只有来自MKp群体的细胞能够发射丙酸盐，这表明MKp群体的成熟阶段更为晚期。骨盆骨的使用急剧增加到细胞数，而两步磁耗竭提高了谱系耗竭的功效。该协议允许随后培养单细胞的MEP和MKp群体，这些细胞与转录组学和蛋白质组学分析一起，肯定必须破译所涉及的机制，实施生物发生。

Research

JoVE Journal

Biology

Mouse Adrenal Chromaffin Cell Isolation

鼠标肾上腺嗜铬细胞分离

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

科研

生物学

Mouse Dorsal Forebrain Explant Isolation

鼠标背的前脑植隔离

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation of Mouse Salivary Gland Stem Cells

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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

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 ...

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 ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...