Name: preparation of whole bone marrow for mass cytometry analysis of neutrophil-lineage cells
Uploaded: 2019-06-19T14:30:00
Description: Watch this Scientific Journal Video about Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells at JoVE.com

我们要感谢LJI流式细胞学核心协助进行大规模细胞测量程序。这项工作得到了NIH授予R01HL134236、P01HL136275和R01CA202987(全部授予C.C.H)和ADA7-12-MN-31(04)(C.C.H.和Y.P.Z)的支持。

所有实验均遵循拉霍亚过敏和免疫学研究所动物护理和使用委员会批准的准则,并根据国家卫生研究院的《实验室动物护理和使用指南》。
1. 收获小鼠骨髓 (BM)
<ol><li>从商业供应商处购买 C57BL/6J 鼠标。在无病原体的设施中,在微型隔离器笼中喂养标准的啮齿动物食物,并饲养房屋。</li>
	<li>使用6-10周大的雄性小鼠进行实验。通过CO2吸入安乐死,然后是宫颈脱位。</li>
	<li>?...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+Early+Unipotent+Neutrophil+Progenitor+with+Pro-tumoral+Activity+in+Mouse+and+Human+Bone+Marrow.">Identification of an Early Unipotent Neutrophil Progenitor with Pro-tumoral Activity in Mouse and Human Bone Marrow.</a> Cell Reports. 24, 2329-2341 (2018).</li><li>Van der Maaten, L. J. P., Hinton, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+High-Dimensional+Data+Using+t-SNE.">Visualizing High-Dimensional Data Using t-SNE.</a> Journal of Machine Learning Research. 9, 2579-2605 (2008).</li><li>Amir, E. A. D., 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=viSNE+enables+visualization+of+high+dimensional+single-cell+data+and+reveals+phenotypic+heterogeneity+of+leukemia.">viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.</a> Nature Biotechnology. 31, 545-552 (2013).</li><li>van der Maaten, L., Hinton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+data+using+t-SNE.">Visualizing data using t-SNE.</a> Journal of Machine Learning Research. 9 (85), 2579-2065 (2008).</li><li>Cloos, 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=Comprehensive+Protocol+to+Sample+and+Process+Bone+Marrow+for+Measuring+Measurable+Residual+Disease+and+Leukemic+Stem+Cells+in+Acute+Myeloid+Leukemia.">Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia.</a> Journal of Visualized Experiment. 133, 56386 (2018).</li><li>Bendall, S. 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在过去的几十年里,荧光流细胞学被用作研究细胞谱系和异质性1、2、3的主要方法。虽然流式细胞学提供了多维数据,但这种方法受到参数选择和光谱重叠的限制。为了克服流式细胞学的弱点,我们利用了CyTOF,它使用重金属同位素代替荧光水,来标记抗体,消除检测通道之间的串扰或细胞的自荧光,因此,可以测量许多在单细胞水平上?...

在过去的几十年中,细胞学方法一直是研究BM造血系统的有力工具。这些方法包括荧光活化流细胞测定法和采用重金属标记抗体的CyTOF新方法。通过鉴定其独特的表面标记表达特征,它们在异质生物标本中发现了许多细胞类型。增加的频谱重叠与更多的通道相关,导致荧光激活流细胞学应用中的数据误差更高。因此,为了丰富荧光激活流细胞学分析感兴趣的细胞群,通常会去除不需要的细胞。例如,Ly6G(或Gr-1)和CD11b被视为成熟的骨髓细胞标记,Ly6G= (或Gr-1+) 和CD11b+细胞在流式细胞测定分析之前,通常使用磁性浓缩试剂盒从BM样品中取出。造血干细胞和祖细胞(HSPCs)或将这些标记物组合在一个转储鸡尾酒通道1,2,3。另一个例子是,中性粒细胞经常从人类血液标本中取出,以丰富外周血单核细胞(PBMC),用于免疫学研究。然而,从小鼠或人类分离出的整个骨髓很少被完整地研究为细胞学分析。
最近,CyTOF已成为研究造血系统4,5,6的革命性工具。使用CyTOF,荧光度标记抗体被重元素报告标记抗体所取代。此方法允许同时测量 40 多个标记,而不必担心频谱重叠。它使完整的生物标本能够进行分析,无需预消耗步骤或转储通道。因此,我们可以从传统的二维流式细胞学图中,以高含量的维数全面地观察造血系统。过去在消耗或浇注过程中省略的细胞群现在可以使用CyTOF4,5生成的高维数据来揭示。我们设计了一个抗体面板,同时测量造血系统中的39个参数,重点是骨髓性linage7。与传统流式细胞测量数据相比,CyTOF生成的前所未有的单细胞高维数据的解释和可视化具有挑战性。计算科学家已经开发了用于高维数据集可视化的维数缩减技术。在本文中,我们使用使用 t 分布随机邻域嵌入 (t-SNE) 技术的算法 viSNE 来分析 CyTOF 数据,并在二维地图上显示高维结果,同时保护高维结构数据8,9,10。在 tSNE 图中,类似的单元格被聚入子集,颜色用于突出显示单元格的特征。例如,在图1中,骨髓细胞根据CyTOF(图1)4产生的33个表面标记的表达模式的相似性,分布到多个细胞集中。在这里,我们研究了小鼠骨髓与我们之前报告的39标记CyTOF面板通过viSNE分析7。viSNE分析我们的CyTOF数据显示一个不明的细胞群,显示HSPC (CD117+)和嗜中性粒细胞 (Ly6G+) 特性 (图2)7。
最后,我们提出了一个方案,用于CyTOF分析处理新鲜的全骨髓。在本文中,我们以小鼠骨髓为例,而此协议也可用于处理人类骨髓样本。协议中也注明了人类骨髓样本的细节。该协议的优点是,它包含一些细节,如孵育时间和温度,经过优化,以保存整个骨髓中的嗜中性粒细胞,以便对完整的整个骨髓进行调查。对于荧光激活流式细胞学应用,此协议也可轻松修改。

<table>
	<tbody>
		<tr>
			<td>CyTOF Antibodies (mouse)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 (Clone 30-F11) -89Y</td>
			<td>Fluidigm</td>
			<td>Cat# 3089005B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human/Mouse CD45R/B220 (Clone RA36B2)-176Yb</td>
			<td>Fluidigm</td>
			<td>Cat# 3176002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD105 (Clone MJ7/18)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 120402; RRID:AB_961070</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD115 (CSF-1R) (Clone AFS98)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 135502; RRID:AB_1937293</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD117/c-kit (Clone 2B8)-166Er</td>
			<td>Fluidigm</td>
			<td>Cat# 3166004B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD11a (Clone M17/4)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 101101; RRID:AB_312774</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11b (Clone M1/70)-148Nd</td>
			<td>Fluidigm</td>
			<td>Cat# 3148003B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11c (Clone N418)-142Nd</td>
			<td>Fluidigm</td>
			<td>Cat# 3142003B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD127 (IL-7R&#945;) (Clone A7R34)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 133919; RRID:AB_2565433</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD150 (Clone TC1512F12.2)-167Er</td>
			<td>Fluidigm</td>
			<td>Cat# 3167004B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD16.2 (Fc&#947;RIV) (Clone 9E9)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 149502; RRID:AB_2565302</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD162 (Clone 4RA10 (RUO))-Purified</td>
			<td>BD Biosciences</td>
			<td>Cat# 557787; RRID:AB_647340</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD169 (Siglec-1) (Clone 3D6.112)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 142402; RRID:AB_10916523</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD182 (CXCR2) (Clone SA044G4)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 149302; RRID:AB_2565277</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD183 (Clone CXCR3-173)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 126502; RRID:AB_1027635</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD335 (NKp46) (Clone 29A1.4)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 137625; RRID:AB_2563744</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD34 (Clone MEC14.7)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 119302; RRID:AB_345280</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD41 (Clone MWReg30)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 133919; RRID:AB_2565433</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD43 (Clone S11)-146Nd</td>
			<td>Fluidigm</td>
			<td>Cat# 3146009B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD48 (Clone HM48.1)-156Gd</td>
			<td>Fluidigm</td>
			<td>Cat# 3156012B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD62L (Clone MEL-14)-MaxPar Ready</td>
			<td>ThermoFisher</td>
			<td>Cat# 14-1351-82; RRID:AB_467481</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD71 (Clone RI7217)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 113802; RRID:AB_313563</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD90 (Clone G7)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 105202; RRID:AB_313169</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse F4/80 (Clone BM8)-159Tb</td>
			<td>Fluidigm</td>
			<td>Cat# 3159009B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Fc&#949;RI&#945; (Clone MAR-1)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 134321; RRID:AB_2563768</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse GM-CSF (MP1-22E9 (RUO))-Purified</td>
			<td>BD Biosciences</td>
			<td>Cat# 554404; RRID:AB_395370</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse I-A/I-E (Clone M5/114.15.2)-174Yb</td>
			<td>Fluidigm</td>
			<td>Cat# 3174003B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ki67 (Clone B56 (RUO))-Purified</td>
			<td>BD Biosciences</td>
			<td>Cat# 556003; RRID:AB_396287</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ly-6A/E (Sca-1) (Clone D7)-169Tm</td>
			<td>Fluidigm</td>
			<td>Cat# 3169015B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ly6B (Clone 7/4)-Purified</td>
			<td>abcam</td>
			<td>Cat# ab53457; RRID:AB_881409</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ly-6G (Clone 1A8)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 127637; RRID:AB_2563784</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse NK1.1 (Clone PK136)-165Ho</td>
			<td>Fluidigm</td>
			<td>Cat# 3165018B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Siglec-F (Clone E50-2440 (RUO))-Purified</td>
			<td>BD Biosciences</td>
			<td>Cat# 552125; RRID:AB_394340</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse TCR&#946; (Clone H57-597)-143Nd</td>
			<td>Fluidigm</td>
			<td>(Clone H57-597)-143Nd</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse TER-119/Erythroid Cells (Clone TER-119)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 116241; RRID:AB_2563789</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals, Peptides and Recombinant Proteins </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody Stabilizer</td>
			<td>CANDOR Bioscience</td>
			<td>Cat# 130050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Cisplatin-194Pt</td>
			<td>Fluidigm</td>
			<td>Cat# 201194</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience 1X RBC Lysis Buffer</td>
			<td>ThermoFisher</td>
			<td>Cat# 00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td>ThermoFisher</td>
			<td>Cat# 00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>EQ Four Element Calibration Beads</td>
			<td>Fluidigm</td>
			<td>Cat# 201078</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>ThermoFisher</td>
			<td>Cat# AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Omega Scientific</td>
			<td>Cat# FB-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Phosphate Buffered Saline solution</td>
			<td>GE Lifesciences</td>
			<td>Cat#SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercalator-Ir</td>
			<td>Fluidigm</td>
			<td>Cat# 201192B</td>
			<td></td>
		</tr>
		<tr>
			<td>MAXPAR Antibody Labeling Kits</td>
			<td>Fluidigm</td>
			<td>http://www.dvssciences.com/product-catalog-maxpar.php</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# 158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA 1X</td>
			<td>Corning</td>
			<td>Cat# 25-053-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental Model: Organism/Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>Stock No: 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Software Alogrithm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bead-based Normalizer</td>
			<td>Finck et al., 2013</td>
			<td>https://med.virginia.edu/flow-cytometry-facility/wp-content/uploads/sites/170/2015/10/3_Finck-Rachel_CUGM_May2013.pdf</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytobank</td>
			<td>Cytobank</td>
			<td>https://www.cytobank.org/</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofkit v1.r.0</td>
			<td>Chen et al., 2016</td>
			<td>https://bioconductor.org/packages/release/bioc/html/cytofkit.html</td>
			<td></td>
		</tr>
		<tr>
			<td>t-SNE</td>
			<td>van der Maaten and Hinton, 2008</td>
			<td>https://cran.r-project.org/web/packages/Rtsne/index.html</td>
			<td></td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

preparation of whole bone marrow for mass cytometry analysis of neutrophil-lineage cells

在本文中,我们提出了一个协议,经过优化,以保存新BM中的中性粒细胞,用于整个BM CyTOF分析。我们使用骨髓偏置的39抗体CyTOF面板,利用该协议对造血系统进行评价,重点是中性粒细胞系细胞。利用开放资源维还原算法viSNE对CyTOF结果进行了分析,并给出了数据,以证明该协议的结果。我们根据这个协议发现了新的嗜中性粒细胞群。这种新鲜全BM制剂的协议可用于1),CyTOF分析,发现来自整个BM的不明细胞群,2),调查整个BM缺陷的血液疾病患者,如白血病,3,协助优化荧光激活流式细胞仪协议,利用新鲜的全BM。

图1作为CyTOF实验的一个示例。在此 tSNE 图中,跨多个小鼠组织的细胞根据 33 参数 CyTOF 面板测量的表面标记表达式配置文件的相似性聚集到子中。具有更多相似属性的细胞根据每个细胞上的 33 个标记的表达式自动聚集在一起,如嗜中性粒细胞、巨噬细胞或 DC。
图2是使用本研究提出的协议进行的小鼠BM CyTOF实验的一个示例结果。该协议...

Watch this Scientific Journal Video about Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells at JoVE.com

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

在这里,我们提出一个协议,处理从小鼠或人类分离的新鲜骨髓(BM),用于对中性粒细胞的超维质量细胞测定(飞行时间,CyTOF)的细胞测定。

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

这种质量细胞学方案保留了整个骨髓的完整性，允许抢救在常规样品制备过程中丢失的信息，以便对研究不足的细胞进行分析。这种快速和毫不费力的骨髓分离方案有助于获得可行的短寿命骨髓细胞群，如罕见的嗜中性粒细胞系子集。使用大规模细胞学来识别以前被低估的免疫细胞，如嗜中性粒细胞-血统细胞可能对各种疾病有广泛的影响。该协议保留了骨髓中存在的短寿命骨髓细胞的完整性，并可以很容易地应用于其他组织类型，如实体肿瘤。我们简化为协议，使其尽可能用户友好，但作为在所有生物研究中，它可能需要一些实践运行来管理。首次这样做时，您可以先准备好所有试剂，然后严格遵守协议。如果您遇到任何问题，您可以连接到 CyTO 论坛，并与可以帮助故障排除的其他 CyTOF 用户交谈。对于生物样品制备，简单的技巧可以产生巨大的差异与最终结果。对于新用户来说，可视化演示更容易掌握协议。如果您是第一次这样做，您可以先准备好您的试剂，然后严格遵守协议。对于骨髓收获，将一只6至10周大的C57黑色六只小鼠放在无菌手术垫上，使用70%乙醇对腹部和后肢进行消毒。使用一双解剖手术剪刀打开腹腔，取出皮肤露出后肢。使用一对钝尖敷料钳，将鼠标头骨放在脚踝下方，并使用一对弯曲的修整钳来稳定钝尖敷料钳下方的头骨。使用钝尖敷料来打破头骨并取出肌肉以露出骨骼。在将头骨置于冷 PBS 中之前，将稳定弯曲的修整钳移动到股骨上，并将钝倾斜敷料滑到膝盖关节下方以握住膝盖。轻轻拉起，使膝盖脱位，将肌肉从膝盖上取下，露出股骨。用弯曲的修整钳抓住暴露的股骨，并使用手术剪刀将股骨从骨骼底部切开。然后将股骨放在 PBS 中。接下来，使用 18 量针在 0.5 毫升微型离心管中打孔，将两个骨骼放在管中，骨骼的开端朝下进入孔中。将 0.5 毫升管放入 1.7 毫升微型离心管中，并在微型离心机中旋转双层管。在离心结束时，确认骨髓已被提取到管的底部。要染色骨髓细胞进行大规模细胞切除术，在室温下将骨髓重新悬浮在一毫升红细胞解液缓冲液中10分钟。在孵育结束时，通过离心收集细胞，并在一毫升冷PBS中重新悬浮颗粒。通过70微米滤网将细胞过滤成15毫升的圆锥管，用9毫升的新鲜冷PBS清洗细胞。将骨髓细胞颗粒重新悬浮在10毫升新鲜、冷的PBS中，进行计数，将五倍10的骨髓细胞等分转移到新的15毫升管中。通过离心收集细胞，将细胞重新悬浮在一毫升质量细胞测量染色缓冲液中，辅以125纳米摩尔顺铂。在室温下五分钟后，将细胞用四毫升新鲜质量的细胞测量缓冲液清洗。将颗粒重新悬浮在50微升的FC受体阻断溶液中。在4摄氏度下10分钟后，将50微升的抗体混合物加入细胞中，轻轻移液混合。不同孵化步骤的温度对于保持骨髓细胞的生存能力非常重要。在4摄氏度下30分钟后，每次洗涤两毫升新鲜质量细胞热缓冲液中清洗细胞，然后将细胞重新悬浮在新鲜准备的1.6%甲醛中，在室温下15分钟。在孵化结束时，通过离心收集细胞，将颗粒重新悬浮在一毫升固定过长缓冲液中，并辅以125纳米摩尔间分液，在4摄氏度下进行隔夜孵育。在通过质量细胞学分析细胞之前，轻轻涡流和离心细胞悬浮液，然后用两毫升新鲜质量细胞学染色缓冲液清洗细胞。接下来，每次洗涤一毫升蒸馏水两次，在第二次洗涤后小心吸升液，然后将细胞重新悬浮在每毫升质量细胞测量缓冲液浓度的1倍10至第六细胞，用于大规模细胞测量分析。在此 T 分布式随机邻域嵌入图中，根据由 33 参数质量细胞学面板测量的表面标记表达式配置文件的相似性，将多个小鼠组织的细胞聚类到子集中。具有更多相似属性的单元格根据每个单元格上的这些标记的表达式自动聚聚在一起。使用这种方法，保留了整个骨髓的完整性，导致发现一个以前未知的细胞群体，同时共同表达嗜中性粒细胞和造血干细胞和祖细胞特征表面标记。由于Ly6G正细胞耗竭，这种细胞群体在骨髓祖体研究中先前被省略，表现出明显的表面标记表达模式。更重要的是，这些数据导致发现了Ly6G正细胞簇的一小部分，该组集没有表达Ly6G，而是与CD117阳性Ly6G阳性细胞紧密聚类，这表明这些细胞与中性粒细胞谱系的相似性，基于该大规模囊细胞学实验中使用的33个表面标记的表达。然后可以建立一个13色荧光激活细胞分拣面板，通过流式细胞学分离中性粒细胞，进行下游功能测定。重要的是尽快执行骨髓分离程序，并在染色过程中将细胞保持在摄氏四度。

Research

JoVE Journal

Immunology and Infection

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

科研

免疫与感染

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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

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 ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

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

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 ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

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

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 ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...