这项议定书是利用在雪松-西奈医学中心的理事会再生医学研究所 (HSG) 的资金, 在美国位免疫学家协会 (HSG) 的免疫学奖学金的职业生涯, 以及学者奖美国血液学学会 (哎)。我们感谢雪松-西奈医疗中心的流式细胞术核心, 以协助进行外地分类。

这里描述的所有方法都是由雪松-西奈医疗中心的机构动物护理和使用委员会 (IACUC) 批准的。
1. 小鼠骨髓的分离和单细胞悬浮液的制备
<ol><li>按照机构指南弄死鼠标。</li>
	<li>将安乐死的老鼠放在背部, 用70% 乙醇 (乙醇) 喷洒。用锋利的尖剪刀在脚踝的每个后肢上做一个小 (3-5 毫米) 切口, 然后将皮肤拉向身体, 从腿部取出, 露出肌肉和骨骼。</li>
	<li>从骨骼中取出肌肉 (四头肌, 腿筋等), 用剪刀修剪, 跟随骨骼的方向。小心不要损坏骨头。</li>
	<li>把股骨从髋关节上脱臼, 切断结缔组织和肌肉, 把腿分开, 小心不要切成骨头。</li>
	<li>通过脱臼脚踝从胫骨中取出脚, 将骨骼放在不育的 PBS 或培养基管中, 然后将它们运到组织培养室。 注: 对于其他一些应用 (例如,从总骨髓细胞中提取大鼠), 可以将骨骼留在冰上一段时间, 但对于祖细胞来说, 要尽快进行, 以避免下调重要的表面标记 (特别是 CD115)。</li>
	<li>在生物安全柜中, 用纸巾擦拭骨头中残留的软组织。 注: 本议定书应在生物安全柜内进行, 如果细胞培养或体内化验后需要无菌样本进行分类。如果不需要无菌细胞, 整个过程就可以在实验室的工作台上进行。</li>
	<li>简单地浸泡在70% 乙醇的骨骼, 以消毒他们, 然后在无菌 PBS 洗。</li>
	<li>将股骨从胫骨和腓骨分开, 通过膝盖切开, 并丢弃腓骨。</li>
	<li>用锋利的剪刀切断骨头的末端。</li>
	<li>用冷不育 PBS 采集骨髓, 直至骨骼呈白色:<ol>
<li>用镊子夹住每根骨头, 并插入一个26G 针连接到一个10毫升的注射器, 其中含有冷不育 PBS 到骨轴一端。</li>
		<li>把骨头放在50毫升离心管上面, 通过骨头冲洗 2-5 毫升的 PBS 以收获骨髓。</li>
		<li>每只老鼠 (2 股骨和2胫骨) 从所有4块骨头中收获骨髓。</li>
	</ol>
</li>
	<li>用1毫升的吸管轻轻地吸管, 将骨髓分解成细胞悬浮。</li>
	<li>通过30微米尼龙网过滤骨髓悬浮物, 消除骨块和细胞团聚体, 从而产生单个细胞悬浮。</li>
	<li>离心机的单细胞悬浮在 280 x g 5 分钟, 并并用重悬细胞颗粒在无菌染色缓冲 (PBS + 0.5% 血清 + 2 毫米 EDTA), 使用5毫升染色缓冲器每只老鼠 (例如, 10 毫升的细胞汇集从股骨和胫骨2小鼠)。</li>
	<li>采取 10 ul 整除的样品, 混合它与 10 ul 台盼蓝, 并加载 10 ul 的结果, 台盼蓝标记的样品上的 hemocytometer。用光显微镜计算细胞数。 注: 如果单元格过于集中以进行可靠计数, 请在添加台盼蓝之前稀释样品。</li>
</ol>2. 磁活化细胞分选 (mac) 的前驱富集
<ol><li>离心机整个单细胞悬浮 (或尽可能多的细胞, 以获得期望的产量) 在 280 x g 5 分钟在4°c 和并用重悬细胞颗粒在 40 ul 染色缓冲 (PBS + 0.5% 血清 + 2 毫米 EDTA) 每 107细胞。 注意: 还可以使用来自沿袭损耗套件制造商 (见材料表) 的 mac 缓冲器作为染色缓冲器的替代品。</li>
	<li>按照沿袭损耗套件提供的说明执行沿袭损耗。下面的说明是在材料表中看到的套件。如果使用了不同的工具包, 请按照制造商的说明进行操作, 然后继续执行步骤2.3。<ol>
<li>添加 10 ul 生物素抗体鸡尾酒每 107细胞, 混合吹打和孵化10分钟在4摄氏度。</li>
		<li>添加 30 ul 染色缓冲/mac 缓冲每 107细胞和混合。</li>
		<li>添加 20 ul 抗生物素微珠每 107细胞。混合好, 孵化15分钟, 在4摄氏度。 注: 在将微珠加入到细胞中之前, 将其涡流, 以确保它们完全分散。</li>
		<li>添加1毫升染色缓冲/mac 缓冲每 107细胞, 离心细胞在 280 x g 为5分钟, 并并用重悬细胞颗粒在 500 ul 染色缓冲/mac 缓冲区多达 108个细胞。对于超过 108细胞, 相应地放大染色缓冲器/mac 缓冲器的体积。</li>
		<li>根据制造商的说明准备自动磁选机。</li>
		<li>将包含标记单元格的管放置到分隔符中, 然后选择负选择程序。</li>
		<li>收集负分数, 其中包含祖丰富的血统阴性 (林) 细胞.放弃正分数, 其中包含磁性标记的谱系阳性细胞。</li>
	</ol>
</li>
	<li>将林细胞在 280 x g 上离心为5分钟, 并用重悬每只老鼠的2毫升染色缓冲/mac 缓冲液中的细胞颗粒 (例如,如果骨髓从2只老鼠中汇集, 则4毫升)。 注: 颗粒现在应该是白色的而不是红色的, 因为红细胞已经耗尽。</li>
	<li>采取 10 ul 整除的林细胞,混合它与 10 ul 台盼蓝, 并加载 10 ul 的结果, 台盼蓝标记的样品上的 hemocytometer。用光显微镜计算细胞数。 注: 如果单元格过于集中以进行可靠计数, 请在添加台盼蓝之前稀释样品。</li>
</ol>3. 用荧光活化细胞分类法进行髓样祖鉴定和分离
<ol><li>标签9离心 (1.5 或2毫升) 管: 1) 无瑕细胞, 2-8) 细胞单一染色荧光共轭抗体对 FcγR (CD16/32), c 套件, Sca-1, CD34, Flt3 (CD135), Ly6C 和 CD115 的电压选择和颜色补偿, 和 9)样本细胞被染色的所有7抗体的祖鉴定和排序。</li>
	<li>将10万个细胞分配给每个控制管 (管 1-8), 所有剩余的细胞 (或更少如果需要) 到样品管 (管 9)。 注: 如果样品体积大于 1.5-2 毫升, 可能需要将样品细胞分布到15毫升管中, 将其离心并并用重悬颗粒 (步骤 3.3), 然后将细胞转移到离心管中, 随后染色步骤。</li>
	<li>将细胞在 1500 x g 处离心5分钟, 并并用重悬 100 ul 染色缓冲器 (PBS + 0.5% 胎体 + 2 毫米 EDTA) 和样品管颗粒在 100 ul 染色缓冲器每 5 x 106个细胞 (如200 x 1 10 细胞的 ul) 控制管丸 。</li>
	<li>添加2微克/毫升 anti-CD16/CD32-APC-Cy7 到 FcγR 单染色管和样品管。涡旋轻轻地混合, 在4摄氏度孵育10分钟。 注意: 重要的是要染色样品细胞与 FcγR (CD16/32) 抗体之前添加其他抗体, 以防止竞争由于非特定 Fc 介导的抗体结合。不要用 FcγR 阻断试剂孵化细胞。</li>
	<li>将其他抗体添加到相应的单一染色管 (每1抗体) 和取样管 (所有6抗体):10 微克/毫升 c 套件-太平洋蓝, 2 微克/毫升 Sca-1-PE-Cy7, 25 微克/毫升 CD34-FITC, 10 微克/毫升 Flt3-APC, 2 微克/毫升 Ly6C PerCP-Cy5.5, 4 微克/毫升 CD115-PE。轻轻地涡旋, 在4摄氏度孵育15分钟。</li>
	<li>将 900 ul 染色缓冲器添加到控制管中, 将每 107个细胞的1毫升染色缓冲到样品管。离心细胞在 1500 x g 5 分钟和并用重悬细胞颗粒在染色缓冲: 200 ul 每控制管和 500 ul 每 25 x 106细胞在样品管。将悬浮细胞转移到5毫升的流管。 注: 如果样品体积大于 1.5-2 毫升, 则可能需要将细胞转移到15毫升管中进行离心。</li>
	<li>根据制造商的指示, 准备流式细胞仪 (分析仪或分拣机)。</li>
	<li>通过流式细胞仪运行无瑕和单染色控制单元, 设置电压和颜色补偿。</li>
	<li>运行示例单元格, 浇下下面概述的祖子集, 并在图 2中进行总结, 如果需要, 则执行该操作的分类以隔离相关的祖子集。 注意: 通常需要获得大约20万个细胞, 以获得至少1000个事件在每个祖门。<ol>
<li>创建所有单元格的点图, 显示 FSC a (x 轴) 和 SSC a (y 轴) 和门, 以排除碎片和死细胞 = > "活" 细胞。</li>
		<li>创建一个 "活" 单元格的点图, 显示 fsc H (x 轴) 和 fsc W (y 轴) 和门, 以排除双峰 = > 活/单线 (FSC) 门。</li>
		<li>创建活/单线 (FSC) 单元格的点图, 显示 ssc H (x 轴) 和 ssc W (y 轴) 和门, 以排除双峰 = > 活/单线 (FSC)/单线 (SSC) 门。</li>
		<li>创建活/单线 (FSC)/单线 (SSC) 单元格的点图, 显示 Sca-1 (x 轴) 和 c 套件 (y 轴), 以及选择 c 套件+ Sca-1 细胞 = > LKS 单元格的门.</li>
		<li>创建 LKS 单元格的点图, 显示 CD34 (x 轴) 和 FcγR (y 轴) 和门以选择 CD34+ FcγRlo细胞 = > "中医", CD34+ FcγRhi细胞 = > "GMPs"。 注意: 必须尽可能准确地将 "中医" 和 "GMPs" 门。使用假彩色密度图或轮廓图来精确的浇口是有帮助的。</li>
		<li>创建一个 "中医" 点图, 显示 CD115 (x 轴) 和 Flt3 (y 轴) 和门, 以选择: Flt3+ CD115螺细胞 = > CMP-Flt3+ CD115lo细胞, Flt3 CD115螺细胞 = > CMP-Flt3 细胞 , Flt3+ CD115hi细胞 = > mdp 以.
</li>
		<li>创建一个 "GMPs" 的点图, 显示 Ly6C (x 轴) 和 FcγR (y 轴) 和门, 以选择 Ly6C 细胞 => "GMP-Ly6C 细胞", Ly6C+细胞 = > "GMP-Ly6C+细胞"。</li>
		<li>创建 "GMP-Ly6C 单元格" 的点图, 显示 CD115 (x 轴) 和 Flt3 (y 轴) 和门以选择: Flt3 CD115螺细胞 = > GMPs.
</li>
		<li>创建 "GMP-Ly6C+单元格" 的点图, 显示 CD115 (x 轴) 和 Flt3 (y 轴) 和门以选择: Flt3- CD115螺细胞 = > GPs, Flt3- CD115hi细胞 = > MPs + cMoPs.
</li>
	</ol>
</li>
</ol>

<ol>
	<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>Y&#225;&#241;ez, A., Ng, M. Y., Hassanzadeh-Kiabi, N., Goodridge, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRF8+acts+in+lineage-committed+rather+than+oligopotent+progenitors+to+control+neutrophil+vs+monocyte+production.">IRF8 acts in lineage-committed rather than oligopotent progenitors to control neutrophil vs monocyte production.</a> Blood. 125 (9), 1452-1459 (2015).</li><li>Auffray, 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=CX3CR1++CD115++CD135++common+macrophage/DC+precursors+and+the+role+of+CX3CR1+in+their+response+to+inflammation.">CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation.</a> Journal of Experimental Medicine. 206 (3), 595-606 (2009).</li><li>Fogg, D. 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=A+clonogenic+bone+marrow+progenitor+specific+for+macrophages+and+dendritic+cells.">A clonogenic bone marrow progenitor specific for macrophages and dendritic cells.</a> Science. 311 (5757), 83-87 (2006).</li><li>Onai, 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=Identification+of+clonogenic+common+Flt3+M-CSFR++plasmacytoid+and+conventional+dendritic+cell+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow.</a> Nature Immunology. 8 (11), 1207-1216 (2007).</li><li>Hettinger, 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=Origin+of+monocytes+and+macrophages+in+a+committed+progenitor.">Origin of monocytes and macrophages in a committed progenitor.</a> Nature Immunology. 14 (8), 821-830 (2013).</li><li>Y&#225;&#241;ez, 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=Granulocyte-monocyte+progenitors+and+monocyte-dendritic+cell+progenitors+independently+produce+functionally+distinct+monocytes.">Granulocyte-monocyte progenitors and monocyte-dendritic cell progenitors independently produce functionally distinct monocytes.</a> Immunity. 47 (5), 890-902 (2017).</li><li>Olsson, 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=Single-cell+analysis+of+mixed-lineage+states+leading+to+a+binary+cell+fate+choice.">Single-cell analysis of mixed-lineage states leading to a binary cell fate choice.</a> Nature. 537 (7622), 698-702 (2016).</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), (2016).</li><li>Y&#225;&#241;ez, A., Goodridge, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon+regulatory+factor+8+and+the+regulation+of+neutrophil,+monocyte,+and+dendritic+cell+production.">Interferon regulatory factor 8 and the regulation of neutrophil, monocyte, and dendritic cell production.</a> Current Opinion in Hematology. 23 (1), 11-17 (2016).</li><li>Manz, M. G., Miyamoto, T., Akashi, K., 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=Prospective+isolation+of+human+clonogenic+common+myeloid+progenitors.">Prospective isolation of human clonogenic common myeloid progenitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (18), 11872-11877 (2002).</li></ol>

作者没有什么可透露的。

小鼠髓质祖细胞魏斯曼门策略1已经是位免疫学家和 hematologists 的黄金标准近20年, 但现在明显的是, "CMP" 和 "GMP" 门是非常异构和更精确需要浇口策略。我们在这里描述的协议允许在 C57BL/6J 小鼠中识别 oligopotent 和沿袭所承诺的子集, 以便更精确地定量化特定的髓样祖细胞和 myelopoiesis 通路的映射, 以及调查在髓系分化的特定阶段运作的分子机制。祖子集可以被排序, 如果需要的话, ?...

单核细胞, 中性粒细胞和树突状干细胞 (DCs) 是由造血祖, 主要是在骨髓中, 由一个称为 myelopoiesis 的过程中产生的髓体细胞。常见的髓样祖细胞 (中医) 有可能产生髓体细胞, 以及巨和红细胞, 而不是淋巴细胞。粒细胞-单核细胞祖 (GMPs) 是由中医衍生而成的, 其产生的是颗粒和单核细胞, 但失去了巨和红细胞电位。单核细胞和经典和浆 DCs (招揽/pDCs) 也被认为是由一般的祖细胞-DC 祖 (mdp 以) 产生的, 这是由中医生产的. 世系势的逐步限制最终导致沿袭前身: 粒细胞祖, 单核细胞祖, 树突状体细胞祖 (图 1)。
魏斯曼和同事报告说, 中医是在林 c-试剂盒+ Sca-1- (LKS) CD34+ FcγR 的小鼠骨髓部分, 而 GMPs 包含在 LKS CD34 + FcγR嗨分数1。然而, 这些 "CMP" 和 "GMP" 分数是非常异构的。例如, "GMP" 分数也包含血统承诺粒细胞祖和单核细胞祖1,2。mdp 以分别被报告为 CX3CR1+ Flt3+ CD115+祖细胞也表达 CD34 和 FcγR3,4。mdp 以产生了 cDC/pDC 产生的共同 DC 祖 (CDPs), 据报道, 它表示较低水平的 c 套件 (CD117), 并没有包括在 LKS 分数5.
以前假定单核细胞是通过单一途径产生的 (CMP-GMP-MDP 单核细胞)。与这个模型相一致的是, 由 GMPs (命名的单核细胞祖, MPs)2和 mdp 以 (命名的共同单核细胞祖, cMoPs) 产生的单核细胞的祖细胞,6在共享表面标记表达式的基础上似乎是同一单元格.然而, 我们最近证明单核细胞是由 GMPs 和 mdp 以独立产生的, 并且能够通过单细胞 RNA 测序7区分 MPs 和 cMoPs。
我们最近修改了魏斯曼 "CMP" 和 "GMP" 浇口战略, 以确定6脂蛋白亚组分的 C57BL/6J 小鼠骨髓含有不同的 oligopotent 和血统承诺髓祖亚群。我们首次报告说, Ly6C 和 CD115 染色允许隔离 oligopotent GMPs, 以及粒细胞祖 (GPs) 和单核细胞祖 (我们目前无法分离的 MPs 和 cMoPs) 从 "GMP" 分数2 (LKS-CD34+ FcγRhi门;图 1)。我们随后证明, mdp 以主要见于 "CMP" 分数 (LKS- CD34+ FcγRlo门), 其中还包含 Flt3+ CD115lo和 Flt3-子集7 (图1).CMP-Flt3+ CD115 的 GMPs 和 mdp 以在收养转移时产生。CMP-Flt3 子集包含的祖细胞似乎是中间体之间的 CMP-Flt3+ CD115 和 GMPs。与 mdp 以不同, CMP-Flt3+ CD115 和 CMP-Flt3 分数也具有巨和红细胞电位。
然而, 必须指出的是, 目前尚不清楚 "CMP" 分数是否包含真正 oligopotent 的祖细胞 (例如, CMP-Flt3+ CD115 中的单个单元, 它拥有中性粒细胞,单核细胞、DC、巨和红细胞电位), 或二者择一地, 构成了一个具有更限制性血统潜能的祖先的混合物。菌落形成检测 (纤维素培养) 揭示了粒细胞 (中性粒细胞), 红细胞, 单核和巨电位 (GEMM 细胞) 在 "CMP", CMP-Flt3+ CD115lo和 CMP-Flt3-分数1 ,7、但不允许对直流电位进行评估。与此相反, 菌落形成检测表明, 在 "GMP" 分数1,2, oligopotent GMPs (祖细胞和单核细胞潜能) 的存在, 这是支持的最近单细胞transcriptomic 分析8。然而, 目前还不知道这些 oligopotent GMPs 是否也产生其他粒细胞 (嗜酸性粒、嗜碱性细胞和肥大的干细胞)。
在这些研究的基础上, 我们现在展示了7表面标记 (c 套件, Sca-1, CD34, FcγR, Flt3, Ly6C 和 CD115) 可以用来识别和分离这6个子集的 oligopotent 和血统承诺的髓细胞祖细胞。这里描述的协议可以应用于体外培养试验 (纤维素或液体培养), 在小鼠体内移植实验, 分子分析 (块状和单细胞 RNA 测序, 西方印迹, 等)。
该协议由3个阶段组成: 1) 制备骨髓细胞单细胞悬浮液, 2) 对造血祖 (磁性活化细胞分选) 和 3) 进行富集鉴定, 并根据需要隔离祖子集, 通过流动细胞术 (根据需要使用分析仪或分拣机)。第一步是从安乐死小鼠的股骨和胫骨中分离骨髓细胞, 类似于先前描述的9项协议。接下来, 通过对红细胞、中性粒细胞、单核细胞、淋巴细胞等细胞表面标志物的抗体, 对茎和祖细胞进行浓缩, 以耗尽分化的细胞。这不是强制性的, 但强烈建议优化检测的祖子集, 并减少所需的抗体数量的祖鉴定和流式细胞术所需的时间。下面的沿袭损耗协议描述了磁活化细胞分类 (mac) 使用鼠标谱系细胞耗尽套件 (其中包含生物素化抗体对 CD5, CD45R (B220), CD11b, Gr-1 (Ly6G/C), 7-4 和 Ter-119, 外加抗生物素微珠) 和自动磁选机。最后一步是流式细胞术对祖子集的识别 (和排序, 如果需要的话)。下面描述的抗体面板 (另见表 1) 已设计用于流式细胞仪 (分析仪或分拣机) 与4激光器 (405 nm, 488 nm, 561 nm, 640 nm)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58061/58061fig1.jpg"> 图 1: 中性粒细胞、单核细胞、DC 祖和分化通路.最近修订的 myelopoiesis7模型被说明, 魏斯曼门为 "中医" (蓝色) 和 "GMPs" (绿色)1覆盖。这一数字已从亚涅斯等20177修改。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58061/58061fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="0" cellpadding="0" cellspacing="0" width="945">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Mouse: Wild-type C57BL/6J (CD45.2)</td>
			<td style="width:177px;">The Jackson Laboratories</td>
			<td style="width:172px;">Cat#JAX:000664</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Lineage Cell Depletion Kit, mouse</td>
			<td style="width:177px;">Miltenyi Biotec</td>
			<td style="width:172px;">Cat#130-090-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD34 (clone RAM34) FITC</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">Cat#553733</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse CD16/CD32 (Fc&#947;R; clone 93) APC-Cy7</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#101327</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse Ly6A/E (Sca-1; clone 108113) PE-Cy7</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#108114</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse CD117 (c-Kit; clone 2B8) Pacific Blue</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#105820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse Ly6C (clone HK1.4) PerCP-Cy5.5</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#128012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD115 (clone AFS98) PE</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#135506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD135 (Flt3; clone A2F10.1) APC</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">Cat#560718</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">CountBright Absolute Counting Beads</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:172px;">Cat#C36950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">AutoMACS Separator</td>
			<td style="width:177px;">Miltenyi Biotec</td>
			<td style="width:172px;">N/A</td>
			<td>Use the &#34;deplete&#34; program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">BD LSRFortessa</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">N/A</td>
			<td>5 lasers, 15 colors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">BD FACS Aria III cell sorter</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">N/A</td>
			<td>5 lasers, 13 colors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">FlowJo</td>
			<td style="width:177px;">FlowJo, LLC</td>
			<td style="width:172px;">https://www.flowjo.com</td>
			<td>For further analysis of the .fcs files</td>
		</tr>
	</tbody>
</table>

identification and isolation of oligopotent and lineage-committed myeloid progenitors from mouse bone marrow

产生中性粒细胞、单核细胞和树突状干细胞 (DCs) 的髓祖原体可以在小鼠骨髓中发现并分离, 用于血液和免疫学分析。例如, 对髓祖种群的细胞和分子特性的研究可以揭示白血病转化的机制, 或者证明免疫系统对病原体暴露的反应。以前描述的流式细胞术策略, 为髓质祖鉴定已在许多领域取得了重大进展, 但他们确定的分数是非常异构的。最常用的浇口策略定义了为所需种群丰富的骨髓成分, 但也含有大量的 "污染" 祖细胞。我们最近的研究已经解决了这种异质性的大部分问题, 我们在这里提出的协议允许从2以前描述的骨髓分数中分离出6亚 oligopotent 和沿袭的髓样祖细胞。该协议描述了3个阶段: 1) 骨髓细胞的分离, 2) 通过磁活化细胞分选 (互连法) 对造血祖体细胞进行浓缩, 3) 流式细胞仪识别髓祖亚群 (包括荧光活化细胞分类, 如有需要, 则进行。这种方法允许祖细胞的数量和分离的各种体外和体内应用, 并已产生了新的洞察力的途径和机制的中性粒, 单核细胞和 DC 分化。

使用上述协议, 有可能获得1亿细胞 (包括红细胞, 或5000万有核细胞) 的股骨和胫骨 (2 条腿) 的一个 C57BL/6J 鼠 (6-8 周, 男性或女性)。1-200万林细胞可以被孤立的每只老鼠的林+细胞耗竭。
6髓系祖群中的每一个都构成了 1- 4% 的林细胞。血统耗尽是有效的消耗分化细胞和丰富的祖细胞, 但林分包含了很...

Watch this Scientific Journal Video about Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow at JoVE.com

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

我们展示了如何用磁性和荧光分选 (mac 和流动力学) 相结合的方法, 从小鼠骨髓中识别和分离出6个髓样祖细胞。本协议可用于体外培养测定 (纤维素或液体培养),体内移植实验, RNA/蛋白质分析。

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 ...

identification-isolation-oligopotent-lineage-committed-myeloid

Research

JoVE Journal

Immunology and Infection

8.4K 次观看.  Cedars-Sinai Medical Center. 我们展示了如何用磁性和荧光分选 (mac 和流动力学) 相结合的方法, 从小鼠骨髓中识别和分离出6个髓样祖细胞。本协议可用于体外培养测定 (纤维素或液体培养),体内移植实验, RNA/蛋白质分析。

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

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

科研

免疫与感染

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...