Name: improving the accuracy of flow cytometric assessment of mitochondrial membrane potential in hematopoietic stem and progenitor cells through the inhibition of efflux pumps
Uploaded: 2019-07-30T15:00:00
Description: Watch this Scientific Journal Video about Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of

作者感谢伊托实验室的所有成员,特别是伊都和H Sato,以及爱因斯坦干细胞研究所的评论和爱因斯坦流细胞测定和分析成像核心设施(由美国国家癌症研究所资助P30 CA013330)为帮助进行实验。K.I. 由美国国家卫生研究院(R01DK98263、R01DK115577和R01DK100689)和纽约州卫生局资助,担任爱因斯坦单细胞基因组学/基因组学(C029154)的核心主任。伊托是白血病和淋巴瘤学会的研究学者。

这里描述的所有方法都已获得阿尔伯特·爱因斯坦医学院机构动物护理和使用委员会(IACUC)的批准。
1. 准备解决方案
<ol><li>染色缓冲液(磷酸盐缓冲盐水(PBS)= 2%胎儿牛血清(FBS):在无菌PBS溶液的500mL中加入10mL的FBS。 注:此溶液可在4°C下在无菌条件下储存至少一个月。在开始以下步骤之前,将此溶液的等分 (50 mL) 放在冰上。</li>
	<li>ACK(氯化铵-钾)溶化缓冲液:在开始手?...

<ol>
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J., Luchsinger, L. L., Corrigan, D. J., Williams, L. J., Snoeck, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye-Independent+Methods+Reveal+Elevated+Mitochondrial+Mass+in+Hematopoietic+Stem+Cells.">Dye-Independent Methods Reveal Elevated Mitochondrial Mass in Hematopoietic Stem Cells.</a> Cell Stem Cell. 21 (6), 725-729 (2017).</li><li>Hall, A. M., Rhodes, G. J., Sandoval, R. M., Corridon, P. R., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+multiphoton+imaging+of+mitochondrial+structure+and+function+during+acute+kidney+injury.">In vivo multiphoton imaging of mitochondrial structure and function during acute kidney injury.</a> Kidney International. 83 (1), 72-83 (2013).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Nicolli, A., Basso, E., Petronilli, V., Wenger, R. M., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+cyclophilin+with+the+mitochondrial+inner+membrane+and+regulation+of+the+permeability+transition+pore,+and+cyclosporin+A-sensitive+channel.">Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, and cyclosporin A-sensitive channel.</a> The Journal of Biological Chemistry. 271 (4), 2185-2192 (1996).</li><li>Vannini, 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=The+NAD-Booster+Nicotinamide+Riboside+Potently+Stimulates+Hematopoiesis+through+Increased+Mitochondrial+Clearance.">The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance.</a> Cell Stem Cell. 24 (3), 405-418 (2019).</li></ol>

线粒体膜电位测量是线粒体分析和评估的基石,线粒体对细胞的代谢状态至关重要。在这里,我们描述了一个通过TMRM染色分析μm的协议。TMRM是一种细胞渗透荧光染料,由于μm在活性线粒体中积累,其各自的水平在细胞外、细胞质和线粒体室之间保持平衡。该协议适用于各种染料,包括四甲胺、乙酯(TMRE)和JC-1。适当的染色条件对于获得准确结果至关重要。这些措施包括:防止光线照射、?...

造血干细胞(HSCs)是自我更新的,多能的,能够产生血液1,2的所有细胞。细胞代谢是HSC维持的关键调节器,与转录因子、内在信号和微环境3、4、5一起。因此,正确控制线粒体功能和质量对HSC维护至关重要。
线粒体膜电位(μm)是线粒体评估的关键参数,因为它直接反映了线粒体的功能,它来自电子传输链中的质子泵送活性平衡和通过F的质子流1/FO ATP 合成酶。这两者都是ADP至ATP8、9氧依赖磷酸化所必需的(取决于基因表达和基质可用性)。利用线粒体室的电子性,开发了各种电位染料来测量μm。其中之一是四甲胺甲基酯高氯酸盐(TMRM),已被广泛用于测量+m通过流动细胞测定在各种细胞10,包括造血干细胞和祖细胞11。
线粒体染料在HSC中必须谨慎使用,但是,因为这些细胞的异种生物排泄泵的高活性可能导致染料挤出12。事实上,线粒体染料(如罗达明123)的挤出已经使研究人员能够分离HSCs13,或者通过利用染料Hoechst Blue和Hoechst Red14的差分挤出来识别HSC"侧群"。15.还表明,Fumitremorgin C,ATP绑定盒式子家族G成员2(ABCG2)传输器的特定阻滞剂,不影响HSPCs16中MitoTracker的染色模式。这些结果公布后,在没有异种生物体排泄泵抑制剂的情况下,使用线粒体染料进行了多项研究,导致人们普遍的印象是,HSC只有少量线粒体,低μm16,17,18.
然而,最近,它证明,Verapamil,一个宽谱抑制剂的排泄泵,显著改变了线粒体染料MitoTracker绿色19的染色模式。这种差异可能是由于Fumitremorgin C对Abcg2具有高度选择性,而HSCs也表示其他运输工具,如Abcb1a(它只对富米震金C敏感度弱)19。我们还报告说,其他线粒体染料,如TMRM、诺尼阿克里丁橙和米托斯特橙(MTO)表现出与Mitotracker Green相同的图案。更重要的是,我们观察到,除了线粒体质量11之外,HSPC的流细胞学模式也反映了它们的μm。
TMRM染料的摄入量严格取决于线粒体的负电荷,但染料的积累在摄入和排空泵20间隙之间保持恒定平衡。HSC和成熟细胞群之间异种生物排泄泵表达的差异会影响这种平衡,并可能导致有偏差的结果。在分析电位染料时,应考虑使用专用抑制剂,如Verapamil。在这里,我们描述了一个改进的协议,用于通过基于TMRM的流式细胞测定进行精确μm测量,该该协议通过使用专用抑制剂纠正异种生物运输者活性。

<table>
	<tbody>
		<tr>
			<td>ACK lysing buffer</td>
			<td>Life Technologies</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-biotin</td>
			<td>BD Bioscience</td>
			<td>553086</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3e-biotin</td>
			<td>Life Technologies</td>
			<td>13-0031-85</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-biotin</td>
			<td>Fischer Scientific</td>
			<td>BDB553782</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8-biotin</td>
			<td>Life Technologies</td>
			<td>13-0081-85</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-biotin</td>
			<td>BD Bioscience</td>
			<td>553309</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19-biotin</td>
			<td>BD Bioscience</td>
			<td>553784</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34-FITC</td>
			<td>eBioscience</td>
			<td>11-0341-85</td>
			<td></td>
		</tr>
		<tr>
			<td>CD48-APC</td>
			<td>eBioscience</td>
			<td>17-0481-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD135-biotin</td>
			<td>eBioscience</td>
			<td>13-1351-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD150-PerCP/Cy5.5&#160;</td>
			<td>Biolegend</td>
			<td>115922</td>
			<td></td>
		</tr>
		<tr>
			<td>c-kit-APC/Cy7</td>
			<td>Biolegend</td>
			<td>105826</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclosporin H</td>
			<td>Millipore Sigma</td>
			<td>SML1575-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI solution (1mg/mL)</td>
			<td>Life Technologies</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Denville</td>
			<td>FB5001-H</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Millipore Sigma</td>
			<td>C2920-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gr1-biotin</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM-biotin</td>
			<td>Life Technologies</td>
			<td>13-5790-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Il7R&#945;-biotin</td>
			<td>eBioscience</td>
			<td>13-1271-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Nk1.1-biotin</td>
			<td>Fischer Scientific</td>
			<td>BDB553163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sca-1-PE/Cy7</td>
			<td>eBioscience</td>
			<td>25-5981-81</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF murine</td>
			<td>PEPROTECH</td>
			<td>250-03-10UG</td>
			<td></td>
		</tr>
		<tr>
			<td>StemSpan SFEM medium</td>
			<td>STEMCELL technologies</td>
			<td>9605</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-Pacific Blue</td>
			<td>eBioscience</td>
			<td>48-4317-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter119-biotin</td>
			<td>Fischer Scientific</td>
			<td>BDB553672</td>
			<td></td>
		</tr>
		<tr>
			<td>TMRM</td>
			<td>Millipore Sigma</td>
			<td>T5428-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>TPO</td>
			<td>PEPROTECH</td>
			<td>315-14-10UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Verapamil hydrochloride</td>
			<td>Millipore Sigma</td>
			<td>V4629-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

improving the accuracy of flow cytometric assessment of mitochondrial membrane potential in hematopoietic stem and progenitor cells through the inhibition of efflux pumps

由于细胞代谢是造血干细胞(HSC)自我更新的关键调节剂,因此HSC研究人员对线粒体在造血平衡中所起的各种作用进行了广泛的研究。线粒体活性水平反映在其膜电位 (μm) 中,可以通过细胞渗透阳离子染料(如 TMRM(四甲基二甲胺、甲基酯)进行测量。然而,排流泵从细胞中挤出这些染料的能力会限制其效用。在评估 HSC 时,由此产生的测量偏差尤为重要,因为异种生物转运剂在 HSC 中表现出比分化细胞更高的表达和活性水平。在这里,我们描述了一个利用Verapamil(一种排泄泵抑制剂)来精确测量多个骨髓群的μm的协议。由此产生的泵活性抑制表明,增加造血干细胞和祖细胞(HSPCs)的TMRM强度,同时在成熟馏分中保持相对不变。这突出表明,在使用_m依赖染料时,需要密切注意染料-排泄物活性,并且根据编写和可视化,此协议可用于准确比较骨髓内的不同种群或同一种群跨越不同的实验模型。

上述协议使 BM-MNC 易于从鼠标模型隔离。图1总结了该方案的主要步骤:骨分离、冲出骨髓、红细胞裂解和抗体染色,然后是TMRM染色,以测量特定造血人群的线粒体膜电位.
BM-MNIC包含若干细胞群,包括HC。该协议中使用的抗体鸡尾酒在HSCs(CD34+和CD34+)、多能祖细胞(MPPs)、Lin+以及Lin+细胞的纯化方面已经成熟,分别为21。 <sup class="xref"...

Watch this Scientific Journal Video about Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of 

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

异种生物排泄泵在造血干细胞和祖细胞(HSPCs)中高度活跃,并导致TMRM(线粒体膜潜在荧光染料)的挤出。在这里,我们提出了一个协议,以准确测量线粒体膜电位在HSPC与TMRM的存在,Verapamil,一个排泄泵抑制剂。

通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

线粒体功能对造血干细胞自我更新至关重要。线粒体活性是内线粒体膜电位的反射器，可以通过TMM（一种细胞荧光、细胞渗透的阳离子染料）进行测量。异生物流泵在 HSC 中高度活跃，并导致 TMRM 挤出。该协议的主要优点是纠正这种效果，利用维拉帕米尔，一个抑制的流水泵。首先将骨髓与小鼠股骨隔离。用一把钳子和锋利的剪刀剪下两只后爪。然后在小鼠的腹皮上做一个小剪贴，然后伸展它。提取股骨和头骨，同时注意不要将股骨的头拔出，将骨骼放在一个六孔板中，里面装满了1.5毫升的染色缓冲液。从骨骼中去除肌肉，切开骨骼的末端，让骨髓退出。将清洁的骨骼放在新井中，用 1.5 毫升染色缓冲液。然后，用三毫升注射器和25针冲洗出骨髓，直到骨头变白。将所有细胞收集在1.5毫升管中，在180倍G下离心5分钟，然后丢弃上一代。小心地将颗粒重新放入300微升的冰冷CK开水缓冲液中，将细胞放在冰上一分钟。然后立即通过添加一毫升染色缓冲液来停用解。将细胞在180倍G.重新暂停的细胞颗粒中离心5分钟，该颗粒应显示为白色，在一毫升染色缓冲液中。使用35微米尼龙网的细胞滤网过滤细胞，获得单核细胞。首先准备血统鸡尾酒，和荧光结合抗体，根据手稿说明。将抗体放在冰上，防止光线。然后，将样品在 180 倍 G 下离心 5 分钟，然后丢弃上一代。加入400微升的血统鸡尾酒，以及4个微升CD135生物素化抗体到细胞颗粒，并漩涡混合物。然后在冰上孵育30分钟。孵育后，通过加入三毫升染色缓冲液清洗样品，在180倍G下旋转5分钟，然后丢弃上经剂。然后加入400微升的抗体溶液，并涡旋混合物。在冰上再孵育30分钟，然后用染色缓冲液重复洗涤。为了准备TMTM染色溶液，添加2.2微升TMTM库存溶液，1.1微升维拉帕米尔，至1.1毫升无血清造血造造培养基，具有血栓波伊汀和干细胞因子。将骨髓重新在TMTM染色溶液的一毫升中，快速旋涡，在37摄氏度下孵育一小时。使用 35 微米尼龙网的细胞滤网盖过滤样品，以避免堵塞流动的环流仪。然后添加一个微升的DAPI，然后继续进行流式细胞测量。运行骨髓样本，并获取至少一百万个事件。然后在太平洋蓝色图中显示活骨髓单核细胞，以识别CD135林阴和林阳性分数。绘制 APC/Cy7 与 PE/Cy7 的林负分数，以识别多能祖先或 MPP 分数。然后绘制 APC 与 PerCP/Cy5.5 的 MPP 分数，以识别造血干细胞或 HSC 分数。显示 FITC 的 HSC 分数（CD34）以将其划分为 CD34 负 HSC 和 CD34 正 HSC 分数。最后获得每个人群的TMM强度。为确保TMTM染色正常工作，在样品中加入一微升FCCP，最终浓度为1毫摩尔，并在37摄氏度下孵育5分钟。然后获取一百万个事件。添加 FCCP 后，应大幅提高 TMRM 强度。该协议已成功用于用TMTM染料量化各种细胞群中的线粒体膜电位。已经证明，PBS和FBS在培养介质中的存在会影响TMM信号强度。因此，在无血清扩张介质中进行TMM染色非常重要。造血干细胞表达挤出TMTM染料的异源性排泄泵的高活性水平。因此，TMRM 配置文件在存在 Verapamil 或 Cyclosporin H 时也会有所不同，它们会阻塞泵。为了验证TMTM染色的准确性，FCCP 可以去极化线粒体，并降低 TMRM 强度。该协议可以很容易地适应研究HSC与不同的线粒体膜电位染料，包括TMR或JC-1。以及其他线粒体染料，如米托跟踪器或米托索克斯。最近才指出了骨髓群体中流血泵不同活动所继承的重要问题。因此，该协议旨在减少以前发现的差异。

Research

JoVE Journal

Biology

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

从胎盘中的造血干细胞的分离和分析

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

科研

生物学

Flow Cytometry Purification of Mouse Meiotic Cells

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

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

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

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

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

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

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

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

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 ...

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

使用 体内 抑制、免疫荧光和流式细胞术对精子细胞中驱动蛋白-7 CENP-E 进行功能评估

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes ...