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>
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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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58061/58061fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table><tbody><tr><td>Mouse: Wild-type C57BL/6J (CD45.2)</td><td>The Jackson Laboratories</td><td>Cat#JAX:000664</td><td></td></tr><tr><td>Lineage Cell Depletion Kit, mouse</td><td>Miltenyi Biotec</td><td>Cat#130-090-858</td><td></td></tr><tr><td>Rat anti-mouse CD34 (clone RAM34) FITC</td><td>BD Biosciences</td><td>Cat#553733</td><td></td></tr><tr><td>Rat anti-mouse CD16/CD32 (Fc&amp;gamma;R; clone 93) APC-Cy7</td><td>BioLegend</td><td>Cat#101327</td><td></td></tr><tr><td>Rat anti-mouse Ly6A/E (Sca-1; clone 108113) PE-Cy7</td><td>BioLegend</td><td>Cat#108114</td><td></td></tr><tr><td>Rat anti-mouse CD117 (c-Kit; clone 2B8) Pacific Blue</td><td>BioLegend</td><td>Cat#105820</td><td></td></tr><tr><td>Rat anti-mouse Ly6C (clone HK1.4) PerCP-Cy5.5</td><td>BioLegend</td><td>Cat#128012</td><td></td></tr><tr><td>Rat anti-mouse CD115 (clone AFS98) PE</td><td>BioLegend</td><td>Cat#135506</td><td></td></tr><tr><td>Rat anti-mouse CD135 (Flt3; clone A2F10.1) APC</td><td>BD Biosciences</td><td>Cat#560718</td><td></td></tr><tr><td>CountBright Absolute Counting Beads</td><td>Thermo Fisher Scientific</td><td>Cat#C36950</td><td></td></tr><tr><td>AutoMACS Separator</td><td>Miltenyi Biotec</td><td>N/A</td><td>Use the &quot;deplete&quot; program</td></tr><tr><td>BD LSRFortessa</td><td>BD Biosciences</td><td>N/A</td><td>5 lasers, 15 colors</td></tr><tr><td>BD FACS Aria III cell sorter</td><td>BD Biosciences</td><td>N/A</td><td>5 lasers, 13 colors</td></tr><tr><td>FlowJo</td><td>FlowJo, LLC</td><td>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

Universidad de Cartagena UDEC

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

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

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

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 ...

科研

发育生物学

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

小鼠髓质前体 32-脑脊液-R 细胞的增殖和分化

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

髓系肿瘤中骨髓微环境的分离和表征

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

作者聚焦:分析小鼠血液系统恶性肿瘤的骨髓微环境 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

识别骨髓增生异常综合征和急性髓系白血病中的骨髓微环境群体

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

癌症研究

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

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

摘要

探索更多视频

此视频中的章节

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

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

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人脐带血衍生CD34的泛骨髓分化^–造血干细胞和祖细胞

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