美国国立卫生研究院资助 R01 CA149976 支持这项工作。

所有程序必须按照机构动物使用和护理指南执行。该程序是为分析小鼠造血干细胞和祖细胞 (HSCs 和高压), 但可以适应其他细胞群体的分析而制定的。
1. 流式细胞仪分离小鼠 HSCs 和高压的研究
<ol><li>收获小鼠骨髓细胞如描述在文献6。 注: 在图 1和图 2所示的数据中, 骨髓是从 C57BL/6J 小鼠身上采集的。</li>
	<li>按照制造商的说明, 使用鼠标沿袭损耗套件执行骨髓细胞的血统耗尽。</li>
	<li>用抗体对细胞表面的标记进行孵化, 如7所述。在图 1和图 2所示的实验中, 使用了对 Sca-1、试剂盒、Flt3 和沿袭 (林) 标记 Mac1、Gr1、CD3e、B220 和 Ter119 的抗体。</li>
	<li>排序 2000-2万林 Sca1+Flt3 细胞 (HSCs ) 或林-Sca1 试剂盒+细胞(hp) 1.5 毫升离心管含有0.1 毫升磷酸盐缓冲盐水 (PBS) 与2% 胎牛血清。 注意: 对于图 1和图 2所示的数据, 细胞使用了一个带有70µM 喷嘴的流式分析仪进行排序。最大采集量为1.4 毫升。</li>
</ol>2. 样品准备
注意: 此步骤非常关键。仔细处理样品。清除上清液时, 要小心不要扰乱细胞颗粒。
<ol><li>离心机的1.5 毫升管包含排序的细胞在摆动斗转子在4°c, 500 x g, 5 分钟. 用吸管和100µL 吸管提示从细胞颗粒中取出所有但大约20µL。不要扰乱球团。<ol>
<li>如果样品包含少于5000个细胞, 并且被排序的样品的总容量少于0.2 毫升, 转移细胞首先到低捆绑0.2 毫升 PCR 管在离心之前。</li>
		<li>如果样品包含少于5000个细胞和容量是超过0.2 毫升, 用离心机在4°c, 500 x g, 为5分钟旋转细胞下来, 并且去除所有, 但200µL 的上清。再将剩余的200µL 中的颗粒细胞重新悬浮, 然后将细胞转移到0.2 毫升的 PCR 管中, 再次旋转。在第二次旋转后从颗粒中除去 20-30 µL, 然后重新悬浮细胞。</li>
	</ol>
</li>
	<li>并用重悬在试管中左上清的细胞。如有必要, 添加额外的 PBS 在2% 的血清/pbs (你也可以使用2% 牛血清白蛋白 (BSA)/pbs, 或 pbs 单独) 获得浓度 5 x104至 5 x 10 细胞/毫升。 注: 使用细胞计数法收集, 以估计用于悬浮细胞的体积。</li>
	<li>使用 2-5 µL 的重新悬浮细胞, 以确定准确的细胞浓度使用自动单元计数器 (按照制造商的指示) 或 hemocytometer9。</li>
	<li>将含有所需细胞数的重悬浮细胞转移到新的1.5 毫升管中。在图 1, 2000, 1000, 或 500 HSCs 或高压的实验被转移了。所需的细胞数取决于由西方印迹获得的信号的强度, 并根据经验确定。使用20µL 或100µL 离心吸管提示进行转移。</li>
	<li>离心机在摆动斗转子中的细胞在4°c, 500 x g, 5 分钟。</li>
	<li>根据制造商的指示, 生成100x 库存解决方案, 在亚砜中溶解蛋白酶体和磷酸酶抑制剂。</li>
	<li>通过稀释制造商提供的相当于蒸馏水的 4x Laemmli 样品缓冲器, 准备 2x Laemmli 样品缓冲器。添加适量的100x 股蛋白酶体和磷酸酶抑制剂, 以获得最终浓度2x 抑制剂在 2x Laemmli 样本缓冲区。 注: 2x Laemmli 样本缓冲区可以提前进行, 但蛋白酶体和磷酸酶抑制剂应立即添加, 然后添加 2x Laemmli 样本缓冲区 (加上抑制剂) 的细胞颗粒溶解细胞, 如以下步骤所述。</li>
	<li>小心地从细胞颗粒中取出部分清液, 并将其与试管中剩余的上清液结合在一起, 添加等量的 2x Laemmli 样品缓冲器加上蛋白酶体和磷酸酶抑制剂以达到500的最终浓度。单元格/µL 在 1x Laemmli 示例缓冲区中。 注意: 例如, 如果您在步骤2.5 中将2000个单元的总体积20µL 转移到一个管, 则在离心单元格 (步骤 2.6) 后, 应从颗粒中移除上清的18µL, 并将剩余的上清的2个µL 添加相等容积 (2 µL) 2x Laemmli 样品缓冲器, 以达到500细胞/µL 在 1x Laemmli 样品缓冲器中的最终浓度。</li>
	<li>并用重悬颗粒以尽快生成裂解液。在这一点上, 样品可以立即电泳通过 SDS 页凝胶, 或可在液体 N2冻结, 并存储在-80 °c, 以供将来使用。</li>
</ol>3. 电泳
<ol><li>将裂解物在95摄氏度或沸水中加热5分钟。</li>
	<li>Electrophorese 1-40 µL 的裂解物 (包含从500高达2万细胞当量) 通过 SDS 页凝胶在100V 使用标准协议10。 注: 在凝胶中装入的裂解液体积是由产生理想信号所需的细胞数决定的, 并取决于感兴趣的蛋白质的丰度和抗体的质量。图 1中的数据获得了2000、1000和500个细胞当量的裂解物。井的宽度应该在3毫米到1.5 毫米的范围内. 1.5 毫米的牙齿可以用剪刀从商业可用的梳子上剪下来。</li>
</ol>4. 转移和阻止
注: 执行11标准协议后的西方污点。下面简要介绍了这些步骤:
<ol><li>预处理氟 (PVDF) 膜与甲醇十年代, 然后简要地洗涤膜与蒸馏水。</li>
	<li>按照转移器具制造商提供的说明书中的说明, 将蛋白质从 SDS 页凝胶转移到膜上。</li>
	<li>转移后, 2 毫升5% 牛血清白蛋白 (BSA) 在 PBST (PBS 与0.1% 补体) 一夜间在4摄氏度以下标准协议11。</li>
</ol>5. 抗体标记
<ol><li>使用供应商推荐的抗体稀释, 在4摄氏度的情况下, 用抗体在一夜床上孵育细胞膜。 注: 在材料表中, 我们对 HSCs 和高压的抗体以及相应的抗体稀释进行了验证。使用标准程序执行抗体标记8。</li>
</ol>6. 检测
<ol><li>清洗膜3次, 每 PBST 5 分钟室温下清洗。</li>
	<li>室温下用2毫升增强化学发光缓冲剂孵化膜1分钟。根据制造商的说明或显影胶片和胶片处理器, 使用成像系统检测信号。</li>
</ol>

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<li>Signer, R. A., Magee, J. A., Salic, A., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Haematopoietic+stem+cells+require+a+highly+regulated+protein+synthesis+rate%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Haematopoietic stem cells require a highly regulated protein synthesis rate.</a> Nature. 509 (7498), 49-54 (2014).</li>
<li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+differentiation+checkpoint+limits+hematopoietic+stem+cell+self-renewal+in+response+to+DNA+damage%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage.</a> Cell. 148 (5), 1001-1014 (2012).</li>
<li>Hoshii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mTORC1+is+essential+for+leukemia+propagation+but+not+stem+cell+self-renewal%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">mTORC1 is essential for leukemia propagation but not stem cell self-renewal.</a> J Clin Invest. 122 (6), 2114-2129 (2012).</li>
<li>Signer, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+rate+of+protein+synthesis+in+hematopoietic+stem+cells+is+limited+partly+by+4E-BPs%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.</a> Genes Dev. 30 (15), 1698-1703 (2016).</li>
<li>Cai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Runx1+deficiency+decreases+ribosome+biogenesis+and+confers+stress+resistance+to+hematopoietic+stem+and+progenitor+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells.</a> Cell Stem Cell. , (2015).</li>
<li>Loven, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revisiting+global+gene+expression+analysis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Revisiting global gene expression analysis.</a> Cell. 151 (3), 476-482 (2012).</li>
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作者没有利益冲突要申报。

西方印迹是一种常见的技术检测特定的蛋白质和激活信号通路的组织或细胞。通过对通常使用的程序进行小的调整, 我们能够例行地检测15种不同的蛋白质 (材料表), 在 1.5万 HSCs, 在某些情况下, 在 500 HSCs。该协议中最关键的步骤是: 1) 精确计数单元格, 2) 最小化管之间的传输数, 3) 株溶藻单元格直接与 Laemmli 样本缓冲区。当株溶藻的细胞颗粒与 Laemmli 样品缓冲, 我们发现, 而不是删除所有...

造血干细胞 (HSCs) 是自我更新的细胞, 可以引起所有的血血统。它们是骨髓中相对稀少的细胞, 使得生物化学分析变得困难。适合于分析稀有细胞的方法, 如流式细胞术, 对于定量测定细胞表面标记物和胞内蛋白的相对数量非常有用。然而, 细胞内蛋白的分析需要使用细胞通透程序来实现抗体的存取, 而不是所有细胞表面的表位都能存活这些程序1,2。此外, 在不同的蛋白质亚型或裂解产物之间鉴别的抗体通常不能用于流式细胞术, 因此调查人员仍然依靠西方的印迹进行某些类型的分析。
裂解物细胞的印迹分析是大多数实验室的常规程序。细胞可以在保存细胞表面分子表位的本地条件下纯化, 细胞裂解物随后可以被制备和分析。然而, 用西方印迹对稀有的原代细胞种群中的蛋白质进行分析, 可以要求 euthanizing 大量的动物获得足够的细胞。通过对几个步骤进行小的调整, 传统的西方印迹协议能够检测出相对较小数量的 HSCs (500-1.5万, 取决于感兴趣的蛋白质) 的蛋白质。这些调整包括准确计数细胞, 仔细处理细胞颗粒, 减少细胞间的转移, 以最小化细胞损失, 并株溶藻一个定义数量的细胞与集中加载缓冲包含蛋白酶体和磷酸酶抑制剂。许多发表的报告包括西部污点获得与2万或更多 HSCs3,4,5,6,7;这个简单的程序将减少在4到40倍之间产生等效数据所需的细胞和实验动物的数量。该协议旨在以每个单元为基础对结果进行规范化, 而不是内部控制。这使得检测蛋白质水平的全面降低, 如果数据被规范化到内部控制, 可以忽略。本文描述了在细胞基础上规范化的重要性, 用于分析基因表达数据8, 同样的原理也适用于用西方印迹来定量蛋白质。此优化的协议对于需要分析少量单元格的任何人都是有用的。

<table><tbody><tr><td>sodium dodecyl sulfate (SDS)</td><td>Fisher Scientific</td><td>BP166-500</td><td>SDS-PAGE</td></tr><tr><td>TEMED</td><td>Fisher Scientific</td><td>BP150-20</td><td>SDS-PAGE</td></tr><tr><td>30% Acrylamidel Bis solution</td><td>Bio-Rad</td><td>1610158</td><td>SDS-PAGE</td></tr><tr><td>1.5M Tris-HCl pH8.8</td><td>TEKNOVA</td><td>T1588</td><td>SDS-PAGE</td></tr><tr><td>1.0M Tris-HCl pH6.8</td><td>TEKNOVA</td><td>T1068</td><td>SDS-PAGE</td></tr><tr><td>Ammonium Persulfate</td><td>Fisher Scientific</td><td>BP179-25</td><td>SDS-PAGE</td></tr><tr><td>mini-protean 3 comb</td><td>Bio-Rad</td><td>1653360</td><td>SDS-PAGE</td></tr><tr><td>Mini-PROTEAN Tetra Cell</td><td>Bio-Rad</td><td>1658005EDU</td><td>SDS-PAGE</td></tr><tr><td>Transfer System</td><td>Bio-Rad</td><td>1704155EDU</td><td>transfer</td></tr><tr><td>PowerPac HC Power Supply</td><td>Bio-Rad</td><td>1645052EDU</td><td>electrophoresis</td></tr><tr><td>Imaging System</td><td>Bio-Rad</td><td>1708195</td><td>signal detection</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747EDU</td><td>sample preparation</td></tr><tr><td>10x Tris/Glycine/SDS Electrophoresis Buffer</td><td>Bio-Rad</td><td>1610732EDU</td><td>electrophoresis</td></tr><tr><td>2-Mercaptoethanol</td><td>Bio-Rad</td><td>1610710EDU</td><td>sample preparation</td></tr><tr><td>RTA Mini PVDF Transfer Kit, for 40 blots</td><td>Bio-Rad</td><td>1704272</td><td>transfer</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>11697498001</td><td>sample preparation</td></tr><tr><td>Phosphatase Inhibitor Cocktailrs</td><td>Gold Biotechnology</td><td>GB-450-1</td><td>sample preparation</td></tr><tr><td>EIF4G</td><td>Cell signaling</td><td>2469</td><td>WB antibody, validated in 1000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): Fig2</td></tr><tr><td>p-Rps6</td><td>Cell signaling</td><td>4858</td><td>WB antibody,validated in 1000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): Fig2</td></tr><tr><td>actin(HRP conjugated)</td><td>abcam</td><td>ab49900</td><td>WB antibody,validated in 500 cells&lt;br/&gt; antibody concentration: 1:5000&lt;br/&gt; reference(figure): Fig1, Fig2</td></tr><tr><td>LC-3</td><td>Abcam</td><td>ab48394</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig5)</td></tr><tr><td>S6</td><td>Cell Signaling</td><td>2317</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>4EBP1</td><td>Cell Signaling</td><td>9644</td><td>WB antibody, validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>p-4EBP1</td><td>Cell Signaling</td><td>2855</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>TSC2</td><td>Cell Signaling</td><td>4308</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>HSP90</td><td>Cell Signaling</td><td>4877</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig5)</td></tr><tr><td>PTEN</td><td>Cell Signaling</td><td>9188</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>mTOR</td><td>Cell Signaling</td><td>2983</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>p-mTOR</td><td>Cell Signaling</td><td>5536</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>PP2AB</td><td>Cell Signaling</td><td>4953</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>p-AMPKa</td><td>Cell Signaling</td><td>2535</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>actin</td><td>Santa Cruz</td><td>sc-47778</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:3000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>lineage depletion kit (mouse)</td><td>Miltenyi Biotec</td><td>130-090-858</td><td>hematopoietic stem and progenitor cells purification</td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-041-305</td><td>hematopoietic stem and progenitor cells purification</td></tr><tr><td>SCF</td><td>PeproTech</td><td>250-03</td><td>phosphorylation stimulation</td></tr><tr><td>APC-Cy7 c-kit antibody</td><td>ThermoFisher</td><td>A15423</td><td>cell sorting</td></tr><tr><td>anti-B220</td><td>ThermoFisher</td><td>48-0452-82</td><td>cell sorting</td></tr><tr><td>anti-CD3e</td><td>ThermoFisher</td><td>48-0031-82</td><td>cell sorting</td></tr><tr><td>anti-Mac1</td><td>ThermoFisher</td><td>48-0112-82</td><td>cell sorting</td></tr><tr><td>anti-Gr1</td><td>ThermoFisher</td><td>48-5931-82</td><td>cell sorting</td></tr><tr><td>anti-Ter119</td><td>ThermoFisher</td><td>48-5921-82</td><td>cell sorting</td></tr><tr><td>Spacer Plates with 1.0 mm Integrated Spacers</td><td>Bio-Rad</td><td>1653311</td><td>SDS-PAGE</td></tr><tr><td>BSA</td><td>Research Products International</td><td>A30075</td><td>membrane blocking</td></tr><tr><td>serum</td><td>Atlanta Biologicals</td><td>A12450</td><td>medium supplement</td></tr><tr><td>cell counter</td><td>Invitrogen</td><td>AMQAF1000</td><td>cell counting</td></tr><tr><td>hemocytometer</td><td>ThermoFisher</td><td>&amp;nbsp;S17040</td><td>cell counting</td></tr><tr><td>flim</td><td>Denville Scientific</td><td>E3012</td><td>signal detection</td></tr><tr><td>medical film processor</td><td>Konica</td><td>SRC-101A</td><td>signal detection</td></tr><tr><td>Supersignal West Femto Maximum Sensitivity Substrate</td><td>Therom Scientific</td><td>34096</td><td>signal detection</td></tr><tr><td>Allegra X-15R Centrifuge (Rotor SX4750A)</td><td>Beckman Coulter</td><td>X-15R</td><td>sample preparation</td></tr><tr><td>BLUEstain protein ladder</td><td>Gold Biotechnology</td><td>P007-500</td><td>electrophoresis</td></tr><tr><td>cell sorter</td><td>BD</td><td>FACSAria II</td><td>sample preparation</td></tr><tr><td>Incublock</td><td>Denville Scientific</td><td>I0510</td><td>electrophoresis</td></tr></tbody></table>

a western blotting protocol for small numbers of hematopoietic stem cells

造血干细胞 (HSCs) 是罕见的细胞, 与小鼠骨髓中仅含有2.5万表型的长期再生 HSCs。对 HSCs (500-1.5万细胞) 的小数目进行了优化, 并适合于对其进行分析。表型 HSCs 纯化, 准确计数, 直接裂解 Laemmli 样品缓冲。用月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 分析了含有等量细胞的裂解物, 并根据标准的西方印迹协议制备并处理了该印迹。使用此协议, 可以对 2000-5000 HSCs 进行例行分析, 在某些情况下, 数据可以从很少500个单元中获得, 而大多数出版物中报告的2万到4万个单元格。本议定书应普遍适用于其他造血细胞, 并能通过标准实验室程序对少量细胞进行常规分析。

500-2000 纯化 HSCs 和高压钠灯的代表性结果如图 1和图 2所示。图 1中的β肌动蛋白信号可以从从一只老鼠的骨髓中纯化出来的 500 HSCs 和高压钠灯中检测出来。请注意, 将裂解物装入1.5 毫米井, 产生了比加载到3.0 毫米井更强的500高压信号。图 2是 EIF4G 的西方印迹和 Rps6 (p-Rps6) 的磷酸化, ?...

Watch this Scientific Journal Video about A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells at JoVE.com

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

通过对500只造血干细胞或祖细胞进行分析, 优化了标准的西方印迹协议。优化包括仔细处理细胞样本, 限制管道之间的传输, 并直接株溶藻 Laemmli 样本缓冲区中的细胞。

一种用于小数目造血干细胞的西方印迹协议

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

a-western-blotting-protocol-for-small-numbers-hematopoietic-stem

Research

JoVE Journal

Biochemistry

10.3K 次观看.  Cincinnati Children's Hospital Medical Center. 通过对500只造血干细胞或祖细胞进行分析, 优化了标准的西方印迹协议。优化包括仔细处理细胞样本, 限制管道之间的传输, 并直接株溶藻 Laemmli 样本缓冲区中的细胞。

视频: A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

分离和造血干细胞移植（造血干细胞）

科研

生物学

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

Hemoglobinopathies 患者造血干细胞移植后残余供红祖细胞的检测

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

发育生物学

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

造血干细胞代谢分析

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

生物工程

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

通过流式细胞术 同时 评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

免疫与感染

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

流细胞测定方法测量造血干细胞和T细胞的线粒体质量及膜电位

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

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

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 such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

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 ...

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs),&#160;defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

长期和短期造血干细胞的分离方法

Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells ...

一种用于小数目造血干细胞的西方印迹协议

摘要

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分离和造血干细胞移植（造血干细胞）

Hemoglobinopathies 患者造血干细胞移植后残余供红祖细胞的检测

造血干细胞代谢分析

维持静止人类造血干细胞的文化方法

通过流式细胞术同时评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

对于样品制备与TEM分析低细胞数量的另一种方法

流细胞测定方法测量造血干细胞和T细胞的线粒体质量及膜电位

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

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

长期和短期造血干细胞的分离方法

一种用于小数目造血干细胞的西方印迹协议

摘要

探索更多视频

分离和造血干细胞移植（造血干细胞）

Hemoglobinopathies 患者造血干细胞移植后残余供红祖细胞的检测

造血干细胞代谢分析

维持静止人类造血干细胞的文化方法

通过流式细胞术 同时 评估造血干细胞和祖细胞的亲缘关系、分裂数和表型

对于样品制备与TEM分析低细胞数量的另一种方法

流细胞测定方法测量造血干细胞和T细胞的线粒体质量及膜电位

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

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

长期和短期造血干细胞的分离方法

通过流式细胞术同时评估造血干细胞和祖细胞的亲缘关系、分裂数和表型