全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| CyTOF Antibodies (mouse) | |||
| Anti-Mouse CD45 (Clone 30-F11) -89Y | Fluidigm | Cat# 3089005B | |
| Anti-Human/Mouse CD45R/B220 (Clone RA36B2)-176Yb | Fluidigm | Cat# 3176002B | |
| Anti-mouse CD105 (Clone MJ7/18)-Purified | Biolegend | Cat# 120402; RRID:AB_961070 | |
| Anti-mouse CD115 (CSF-1R) (Clone AFS98)-Purified | Biolegend | Cat# 135502; RRID:AB_1937293 | |
| Anti-Mouse CD117/c-kit (Clone 2B8)-166Er | Fluidigm | Cat# 3166004B | |
| Anti-mouse CD11a (Clone M17/4)-Purified | Biolegend | Cat# 101101; RRID:AB_312774 | |
| Anti-Mouse CD11b (Clone M1/70)-148Nd | Fluidigm | Cat# 3148003B | |
| Anti-Mouse CD11c (Clone N418)-142Nd | Fluidigm | Cat# 3142003B | |
| Anti-mouse CD127 (IL-7Rα) (Clone A7R34)-MaxPar Ready | Biolegend | Cat# 133919; RRID:AB_2565433 | |
| Anti-Mouse CD150 (Clone TC1512F12.2)-167Er | Fluidigm | Cat# 3167004B | |
| Anti-mouse CD16.2 (FcγRIV) (Clone 9E9)-Purified | Biolegend | Cat# 149502; RRID:AB_2565302 | |
| Anti-Mouse CD162 (Clone 4RA10 (RUO))-Purified | BD Biosciences | Cat# 557787; RRID:AB_647340 | |
| Anti-mouse CD169 (Siglec-1) (Clone 3D6.112)-Purified | Biolegend | Cat# 142402; RRID:AB_10916523 | |
| Anti-mouse CD182 (CXCR2) (Clone SA044G4)-Purified | Biolegend | Cat# 149302; RRID:AB_2565277 | |
| Anti-mouse CD183 (Clone CXCR3-173)-Purified | Biolegend | Cat# 126502; RRID:AB_1027635 | |
| Anti-mouse CD335 (NKp46) (Clone 29A1.4)-MaxPar Ready | Biolegend | Cat# 137625; RRID:AB_2563744 | |
| Anti-mouse CD34 (Clone MEC14.7)-Purified | Biolegend | Cat# 119302; RRID:AB_345280 | |
| Anti-mouse CD41 (Clone MWReg30)-MaxPar Ready | Biolegend | Cat# 133919; RRID:AB_2565433 | |
| Anti-Mouse CD43 (Clone S11)-146Nd | Fluidigm | Cat# 3146009B | |
| Anti-Mouse CD48 (Clone HM48.1)-156Gd | Fluidigm | Cat# 3156012B | |
| Anti-mouse CD62L (Clone MEL-14)-MaxPar Ready | ThermoFisher | Cat# 14-1351-82; RRID:AB_467481 | |
| Anti-mouse CD71 (Clone RI7217)-Purified | Biolegend | Cat# 113802; RRID:AB_313563 | |
| Anti-mouse CD90 (Clone G7)-Purified | Biolegend | Cat# 105202; RRID:AB_313169 | |
| Anti-Mouse F4/80 (Clone BM8)-159Tb | Fluidigm | Cat# 3159009B | |
| Anti-mouse FcεRIα (Clone MAR-1)-MaxPar Ready | Biolegend | Cat# 134321; RRID:AB_2563768 | |
| Anti-mouse GM-CSF (MP1-22E9 (RUO))-Purified | BD Biosciences | Cat# 554404; RRID:AB_395370 | |
| Anti-Mouse I-A/I-E (Clone M5/114.15.2)-174Yb | Fluidigm | Cat# 3174003B | |
| Anti-Mouse Ki67 (Clone B56 (RUO))-Purified | BD Biosciences | Cat# 556003; RRID:AB_396287 | |
| Anti-Mouse Ly-6A/E (Sca-1) (Clone D7)-169Tm | Fluidigm | Cat# 3169015B | |
| Anti-Mouse Ly6B (Clone 7/4)-Purified | abcam | Cat# ab53457; RRID:AB_881409 | |
| Anti-mouse Ly-6G (Clone 1A8)-MaxPar Ready | Biolegend | Cat# 127637; RRID:AB_2563784 | |
| Anti-Mouse NK1.1 (Clone PK136)-165Ho | Fluidigm | Cat# 3165018B | |
| Anti-Mouse Siglec-F (Clone E50-2440 (RUO))-Purified | BD Biosciences | Cat# 552125; RRID:AB_394340 | |
| Anti-Mouse TCRβ (Clone H57-597)-143Nd | Fluidigm | (Clone H57-597)-143Nd | |
| Anti-mouse TER-119/Erythroid Cells (Clone TER-119)-MaxPar Ready | Biolegend | Cat# 116241; RRID:AB_2563789 | |
| Chemicals, Peptides and Recombinant Proteins | |||
| Antibody Stabilizer | CANDOR Bioscience | Cat# 130050 | |
| Bovine Serum Albumin | Sigma-Aldrich | Cat# A4503 | |
| Cisplatin-194Pt | Fluidigm | Cat# 201194 | |
| eBioscience 1X RBC Lysis Buffer | ThermoFisher | Cat# 00-4333-57 | |
| eBioscience Foxp3 / Transcription Factor Staining Buffer Set | ThermoFisher | Cat# 00-4333-57 | |
| EQ Four Element Calibration Beads | Fluidigm | Cat# 201078 | |
| Ethylenediaminetetraacetic acid (EDTA) | ThermoFisher | Cat# AM9260G | |
| Fetal Bovine Serum | Omega Scientific | Cat# FB-02 | |
| HyClone Phosphate Buffered Saline solution | GE Lifesciences | Cat#SH30256.01 | |
| Intercalator-Ir | Fluidigm | Cat# 201192B | |
| MAXPAR Antibody Labeling Kits | Fluidigm | http://www.dvssciences.com/product-catalog-maxpar.php | |
| Paraformaldehyde | Sigma-Aldrich | Cat# 158127 | |
| Sodium azide | Sigma-Aldrich | Cat# S2002 | |
| Triton X-100 | Sigma-Aldrich | Cat# X100 | |
| Trypsin EDTA 1X | Corning | Cat# 25-053-Cl | |
| Experimental Model: Organism/Strains | |||
| Mouse: C57BL/6J | The Jackson Laboratory | Stock No: 000664 | |
| Software Alogrithm | |||
| Bead-based Normalizer | Finck et al., 2013 | https://med.virginia.edu/flow-cytometry-facility/wp-content/uploads/sites/170/2015/10/3_Finck-Rachel_CUGM_May2013.pdf | |
| Cytobank | Cytobank | https://www.cytobank.org/ | |
| Cytofkit v1.r.0 | Chen et al., 2016 | https://bioconductor.org/packages/release/bioc/html/cytofkit.html | |
| t-SNE | van der Maaten and Hinton, 2008 | https://cran.r-project.org/web/packages/Rtsne/index.html | |
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