搜索

Phenotyping of Live Human PBMC using CyTOFTM Mass Cytometry
采用CyTOF质谱流式细胞仪研究人外周血单核细胞表型   

引用 收藏 2 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Immunity
Jan 2012

Abstract

Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators.

In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOFTM (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.

Keywords: CyTOF (cytof), PBMC (外周血单个核细胞), Phenotyping (表型), Mass cytometry (大量细胞计数法)

Materials and Reagents

  1. Peripheral blood mononuclear cells (PBMC) (fresh, or frozen after Ficoll isolation procedure)
  2. RPMI (HyClone, catalog number: SH30027.01 )
  3. FBS (heat-inactivated before use) (Atlanta Biologicals, catalog number: S11150 )
  4. Pen-Strep-Glutamine (100x) (HyClone, catalog number: SV30082.01 )
  5. Benzonase (25 x 105 U/ml) (Pierce Antibodies, catalog number: 88701 )
  6. MilliQ water [no contact with beakers or bottles washed with soap (due to barium content of most commercial soaps)]
  7. PBS (10x stock) (Rockland, catalog number: MB-008 )
  8. Bovine serum albumin (BSA) (30% w/v in 0.85% NaCl) (Sigma-Aldrich, catalog number: A7284 )
  9. 0.5 M EDTA (pH 8.0) (Hoefer, catalog number: GR123-100 )
  10. Sodium azide (10% w/v solution) (Teknova, catalog number: S0209 )
  11. 2% para-formaldehyde (PFA) (16% w/v aqueous stock, methanol-free, diluted to 2% in 1x CyPBS) (Alfa Aesar, catalog number: 43368 )
  12. Maleimide-DOTA (Macrocyclics, catalog number: B-272 )
  13. 139-Lanthanum (chloride salt) (Sigma-Aldrich, catalog number: 203521 )
  14. 115-Indium (chloride salt) (Sigma-Aldrich, catalog number: 203440 )
  15. Phenotyping antibodies (filtered with 0.1 μm spin filters) (MIllipore, catalog number: UFC30VV00 )
  16. Ir-intercalator stock solution from Fluidigm (catalog number: 201192B ; Rh103-intercalator catalog number: 201103B can be used)
  17. 10x saponin-based permeabilization buffer (eBiosciences, catalog number: 00-8333-56 )
  18. Complete RPMI (see Recipes)
  19. CyPBS (see Recipes)
  20. CyFACS buffer (see Recipes)
  21. Live-dead stain (see Recipes)

Equipment

  1. 37 °C water bath
  2. 96-well plate : 300 μl well volume (250 μl washes) or 1.2 ml well volume (500 μl washes)
    Note: If using a 300 μl plate, do an additional wash step (resuspension and centrifugation) at each point.
  3. Biosafety cabinet
  4. Centrifuge
  5. Calibrated pipettes
  6. ViCell (Beckman Coulter) or Hemocytometer cell counter
  7. CyTOFTM version 1 mass cytometer (Fluidigm Sciences)

Software

  1. FlowJo software
  2. Microsoft Excel

Procedure

  1. Thaw PBMC
    1. Warm Complete RPMI media to 37 °C in water bath. Each sample will require 20 ml of media with benzonase. Calculate the amount needed to thaw all samples, and prepare a separate aliquot of warm media with 1:10,000 benzonase (25 U/ml final concentration; benzonase removes reduces viscosity and background from free DNA from lysed cells). Thaw no more than 10 samples at a time.
    2. Remove samples from liquid nitrogen and transport to lab on dry ice.
    3. Place 10 ml of warmed benzonase media into a 15 ml tube, making a separate tube for each sample.
    4. Thaw frozen vials in 37 °C water bath.
    5. When cells are nearly completely thawed, carry to hood.
    6. Add 1 ml of warm benzonase media from appropriately labeled centrifuge tube slowly to the cells, then transfer the cells to the centrifuge tube. Rinse vial with more media from centrifuge tube to retrieve all cells.
    7. Continue with the rest of the samples as quickly as possible.
    8. Centrifuge cells at (RCF = 483) for 10 min at room temperature.
    9. Remove supernatant from the cells and resuspend the pellet by tapping the tube.
    10. Gently resuspend the pellet in 1 ml warmed benzonase media. Filter cells through a 70 micron cell strainer if needed (i.e. if you observe any clumps). Add 9 ml warmed benzonase media to the tube to make volume 10 ml altogether.
    11. Centrifuge cells (RCF = 483) for 10 min at room temperature.
    12. Remove supernatant from the cells and resuspend the pellet by tapping the tube.
    13. Resuspend cells in 1 ml CyFACS buffer.
    14. Count cells with Vicell (or hemocytometer). To count, take 20 μl cells and dilute with 480 μl CyPBS in Vicell counting chamber. Load onto Vicell as PBMC with a 1:25 dilution factor.
    15. Calculate the resuspension volume needed to obtain 1 million viable cells.
      Note: It is typical to recover 4-8 x 106 cells from a vial frozen at 10 x 106 cells/vial.

  2. Stain cells
    Day one
    1. Add 1 million viable cells from a donor into a well of the 96 well plate. Repeat for all samples. Add CyFACS buffer to approximately 600 μl and centrifuge cells (RCF = 483) for 10 min at room temperature.
    2. Flick or aspirate to remove supernatant, and repeat wash step and centrifugation with 500 μl CyFACS.
    3. Make cocktail in CyFACS buffer of metal-chelating polymer-labeled antibodies according to previously determined titration. Make sufficient volume for each sample to have 50 μl of cocktail. Pipet into 0.1 μm spin filter and centrifuge in a tabletop microcentrifuge (RCF = 14,000) for 10 min at room temperature.
    4. Flick or aspirate to remove supernatant from second wash step B2. Add 50 μl antibody cocktail to each sample. Pipet up and down to mix. Incubate on ice for 60 min.
    5. Add 500 μl CyFACS buffer, then centrifuge cells (RCF = 483) for 10 min at room temperature. Flick or aspirate to remove supernatant, resuspend pellet in 500 μl CyFACS, and centrifuge cells (RCF = 483) for 10 min at room temperature.
    6. Make 1:3,000 dilution in CyPBS of 5 mg/ml live-dead maleimide-DOTA stain. Add 100 μl to each sample, pipetting to mix. Incubate on ice for 30 min.
    7. Add 500 μl CyFACS buffer, then centrifuge cells (RCF = 483) for 10 min at room temperature. Flick or aspirate to remove supernatant, resuspend pellet in 500 μl CyFACS, and centrifuge cells (RCF = 483) for 10 min at room temperature.
    8. Add 100 μl of 2% PFA (in CyPBS); pipet to mix. Place at 4 °C overnight. PFA fixation is required due to permeabilization and MilliQ water wash osmotic stress in Day two.
    Day two
    1. Add 500 μl CyFACS buffer, then centrifuge cells (RCF = 805) for 10 min at 4 °C. Flick or aspirate to remove supernatant, resuspend pellet in 500 μl CyFACS, and centrifuge cells (RCF = 805) for 10 min at 4 °C.
      Note: It is common to increase RCF after fixation, particularly during permabilization steps. Live cells in Day 1 cannot take the stress.
    2. Make 1x saponin permeabilization buffer in CyPBS. Flick or aspirate to remove supernatant from step 9. Add 100 μl of 1x saponin permeabilization buffer to each sample, pipet to mix. Incubate on ice for 45 min.
    3. Add 500 μl CyFACS buffer, then centrifuge cells (RCF = 805) for 10 min at room temperature. Flick or aspirate to remove supernatant, resuspend pellet in 500 μl CyFACS, and centrifuge cells (RCF = 805) for 10 min at 4 °C.
    4. Make 1:2,000 dilution in CyPBS of Ir-intercalator. Add 100 μl of diluted Ir-intercalator solution to each sample, pipet to mix. Incubate at room temperature for 20 min.
    5. Add 500 μl CyFACS buffer, then centrifuge cells (RCF = 805) for 10 min at room temperature. Flick or aspirate to remove supernatant, resuspend pellet in 500 μl CyFACS, and centrifuge cells (RCF = 805) for 10 min at 4 °C. Flick or aspirate to remove supernatant, and repeat at least twice with 500 μl of MilliQ water. The MilliQ water washes are critical to remove buffer salts, which can cause the Current setting on the CyTOF to drift over the course of your experiment.
    6. Acquire samples on the CyTOF, after standard instrument setup procedures. Collect at least one “push” of 500 μl per sample.

  3. Analyze data
    1. Analyze data using FlowJo software using the standard CyTOF template. Adjust gates on a donor-specific basis, if necessary, to control for any differences in background or positive staining intensity. Export the statistics for each gated population to an Excel spreadsheet.

Recipes

  1. Complete RPMI
    Note: Combine and 0.2 μm filter to sterilize before use.
    RPMI 500 ml
    50 ml heat-inactivated FBS
    5 ml Pen-Strep-Glutamine 100x stock
  2. CyPBS
    Dilute 10x PBS to 1x with MilliQ water
  3. CyFACS buffer
    1x CyPBS with 0.1% BSA
    2 mM EDTA and 0.05% Na azide
    Made in MilliQ water
    Note: Do not use FBS!
  4. Live-dead stain
    5 mg/ml maleimide-DOTA loaded with 139-Lanthanum* or 115-Indium*
    *Natural-abundance metal chloride salt used; >95% specified isotope; trace-metal pure 99.99%
    Dissolve solid DOTA-Maleimide at 41.5 mg/ml in L-buffer (Fluidigm) or MilliQ water acidified with nitric acid (100 u 3% v/v nitric acid per 10 ml water) containing 25 mM Metal (III) salt. Incubate at room temperature for 15 min, then aliquot and freeze at -20 °C.
    41.5 mg/ml is 50 mM for the DOTA Maleimide. The 2:1 molar ratio of DOTA: Metal is intended to reduce/eliminate unchelated metal in the barcoding reagent.
    Note: Each new batch of metal-loaded maleimide-DOTA should be titered for best resolution of live vs dead cells, with minimal background. Therefore, final working concentration and final dilution may vary slightly from batch to batch.

Acknowledgments

We would like to acknowledge funding from NIH grants S10RR027582, 5U19AI057229, and 5U19AI090019. We would also like to thank Dr. Evan Newell for initial protocol development (Newell et al., 2012).

References

  1. Maecker, H. T., McCoy, J. P. and Nussenblatt, R. (2012). Standardizing immunophenotyping for the Human Immunology Project. Nat Rev Immunol 12(3): 191-200.
  2. Newell, E. W., Sigal, N., Bendall, S. C., Nolan, G. P. and Davis, M. M. (2012). Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity 36(1): 142-152.

简介

单细胞分析已成为免疫学中重要的一种方法。荧光流式细胞仪一直是主要的参与者。然而,由于诸如自身荧光和不同荧光团之间的发射溢出的问题,正在开发替代技术。近年来,已经出现了大量细胞计数,其中用金属离子标记的抗体通过ICP-MS检测。为了看到电池,必须存在质量窗口中的金属;没有与前向或侧向散射的类似参数。选择的当前质量窗口大约是AW 103-196,其包括用于大多数抗体标记的镧系元素,以及用于DNA嵌入剂的铱和铑。在本方案中,我们使用用MAXPAR金属标记的抗体混合物螯合聚合物对先前冷冻保存的表面染色活的PBMC。许多这些标记取自标准荧光表型组(Maecker等人,2012)。不使用细胞内抗体。我们使用CyTOF TM (通过飞行时间细胞计数)质谱仪获取ICP-MS数据。使用FlowJo软件的双计数信号数据的后续分析允许基于每个质量通道中的双计数信号来分析细胞类型。确定每种细胞类型的百分比,并报告为父细胞类型的百分比。

关键字:cytof, 外周血单个核细胞, 表型, 大量细胞计数法

材料和试剂

  1. 外周血单核细胞(PBMC)(新鲜或在Ficoll分离步骤后冷冻)
  2. RPMI(HyClone,目录号:SH30027.01)
  3. FBS(使用前热灭活)(Atlanta Biologicals,目录号:S11150)
  4. Pen-Strep-Glutamine(100x)(HyClone,目录号:SV30082.01)
  5. Benzonase(25×10 5 U/ml)(Pierce Antibodies,目录号:88701)
  6. MilliQ水[无需接触烧杯或用肥皂洗涤的瓶子(由于大多数商业皂的钡含量)]
  7. PBS(10x储备液)(Rockland,目录号:MB-008)
  8. 将牛血清白蛋白(BSA)(在0.85%NaCl中的30%w/v)(Sigma-Aldrich,目录号:A7284)
  9. 0.5M EDTA(pH8.0)(Hoefer,目录号:GR123-100)
  10. 叠氮化钠(10%w/v溶液)(Teknova,目录号:S0209)
  11. 2%多聚甲醛(PFA)(16%w/v含水储液,不含甲醇,在1x CyPBS中稀释至2%)(Alfa Aesar,目录号:43368)
  12. 马来酰亚胺-DOTA(Macrocyclics,目录号:B-272)
  13. 139-镧(氯化物盐)(Sigma-Aldrich,目录号:203521)
  14. 115-铟(氯化物盐)(Sigma-Aldrich,目录号:203440)
  15. 表型抗体(用0.1μm旋转过滤器过滤)(MIllipore,目录号:UFC30VV00)
  16. 可以使用来自Fluidigm的Ir-嵌入剂储液(目录号:201192B; Rh103-嵌入剂目录号:201103B)
  17. 基于10×皂苷的透化缓冲液(eBiosciences,目录号:00-8333-56)
  18. 完成RPMI(参见配方)
  19. CyPBS(参见配方)
  20. CyFACS缓冲区(参见配方)
  21. 活死皮(见配方)

设备

  1. 37°C水浴
  2. 96孔板:300μl孔体积(250μl洗涤)或1.2ml孔体积(500μl洗涤)
    注意:如果使用300μl板,在每个点进行额外的洗涤步骤(重悬和离心)。
  3. 生物安全柜
  4. 离心机
  5. 校准移液器
  6. ViCell(Beckman Coulter)或血细胞计数器计数器
  7. CyTOF TM 版本1质谱仪(Fluidigm Sciences)

软件

  1. FlowJo软件
  2. Microsoft Excel

程序

  1. 解冻PBMC
    1. 将热完全RPMI培养基在37℃水浴中。 每个样品将 需要20ml的具有benzonase的培养基。 计算所需的金额 解冻所有样品,并制备单独的温热培养基的等分试样 1:10,000 benzonase(25U/ml终浓度; benzonase去除 降低来自裂解细胞的游离DNA的粘度和背景)。 解冻 一次不超过10个样品。
    2. 从液氮中取出样品,并在干冰上运输到实验室
    3. 将10毫升温热的benzonase培养基放入15毫升管,为每个样品制作一个单独的管。
    4. 在37℃水浴中解冻冷冻的小瓶。
    5. 当细胞几乎完全解冻,携带到罩。
    6. 从适当标记加入1毫升温暖benzonase培养基 离心管慢慢地将细胞转移到细胞中 离心管。 用更多的培养基从离心管中冲洗小瓶 检索所有单元格。
    7. 尽快继续其余样品。
    8. 在室温下在(RCF = 483)离心细胞10分钟
    9. 从细胞中除去上清液,并通过轻敲管子重悬沉淀
    10. 轻轻地将沉淀重悬在1ml温热的benzonase培养基中。 过滤 细胞通过70微米的细胞过滤器(如果需要)(即如果你观察 任何团块)。 加入9毫升加热的benzonase培养基到管中,使体积 共计10ml。
    11. 在室温下离心细胞(RCF = 483)10分钟
    12. 从细胞中除去上清液,并通过轻敲管子重悬沉淀
    13. 将细胞重悬在1ml CyFACS缓冲液中
    14. 用Vicell(或血细胞计数器)计数细胞。 计数,取20微升 细胞并在Vicell计数室中用480μlCyPBS稀释。 加载   Vicell作为PBMC,1:25稀释因子
    15. 计算获得1百万活细胞所需的重悬浮体积。
      注意:通常,从冻结在10 x 10的样品瓶中恢复4-8 x 10 6 6

  2. 染色池
    第一天
    1. 将来自供体的100万个活细胞加入96孔的孔中 盘子。 对所有样品重复此操作。 加入CyFACS缓冲液约600μl   并在室温下离心细胞(RCF = 483)10分钟
    2. 轻轻或吸出以除去上清液,并重复洗涤步骤并用500μlCyFACS离心
    3. 在金属螯合聚合物标记的CyFACS缓冲液中制备鸡尾酒 抗体。 充足 每个样品的体积具有50μl的混合物。 移入0.1μm 旋转过滤器并在台式微量离心机中离心(RCF = 14,000) 在室温下10分钟
    4. 轻拂或吸出即可 来自第二洗涤步骤B2的上清液。 加入50μl抗体混合物 每个样品。 吸移上下混合。 在冰上孵育60分钟。
    5. 加入500微升CyFACS缓冲液,然后离心细胞(RCF = 483)10 min。 轻拂或吸出以除去上清液, 在500μlCyFACS中重悬沉淀,并离心细胞(RCF = 483) 室温下10分钟
    6. 在CyPBS中进行1:3,000稀释 mg/ml活死亡马来酰亚胺-DOTA染色。 每个样品加入100μl, 移液混合。 在冰上孵育30分钟。
    7. 加入500微升CyFACS 缓冲液,然后在室温离心细胞(RCF = 483)10分钟 温度。 轻拂或吸出以除去上清液,重悬沉淀 在500μlCyFACS中,并在室温下离心细胞(RCF = 483)10分钟 温度
    8. 加入100μl的2%PFA(在CyPBS中); 移液器混合。 地点   在4℃过夜。 由于透化和需要PFA固定   MilliQ水洗渗透压力在第二天。
    第二天
    1. 加入500微升CyFACS缓冲液,然后离心细胞(RCF = 805)10分钟 在4℃。 轻拂或吸出以去除上清液,将沉淀重悬浮 500μlCyFACS,并在4℃下离心细胞(RCF = 805)10分钟 注意:在固定后,特别是在增加RCF是常见的 永久化步骤。 第1天的活细胞不能承受压力。
    2. 在CyPBS中制备1x皂苷透化缓冲液。 轻弹或吸气 以从步骤9除去上清液。加入100μl的1x皂苷 透化缓冲液加入每个样品,用移液管混合。 在冰上孵化 45分钟。
    3. 加入500微升CyFACS缓冲液,然后离心细胞(RCF   = 805)在室温下10分钟。 轻拂或吸出即可 上清液,在500μlCyFACS中重悬沉淀,并离心细胞 (RCF = 805)在4℃下10分钟
    4. 在CyPBS中进行1:2000稀释 的Ir-嵌入剂。 加入100μl稀释的Ir-嵌入剂溶液 每个样品,移液管混合。 在室温下孵育20分钟。
    5. 加入500微升CyFACS缓冲液,然后离心细胞(RCF = 805)10 min。 轻拂或吸出以除去上清液, 在500μlCyFACS中重悬沉淀,并离心细胞(RCF = 805) 在4℃下10分钟。 轻拂或吸出以去除上清液,并重复 用500μlMilliQ水至少洗涤两次。 MilliQ水洗 关键是要去除缓冲盐,这可能导致电流设置 CyTOF在实验过程中漂移
    6. 在标准仪器设置程序后,在CyTOF上采集样品。 收集至少一个"推"每个样品500μl。

  3. 分析数据
    1. 使用FlowJo软件使用标准CyTOF模板分析数据。 根据捐赠者特定的基础调整门,如果必要,控制任何   背景差异或阳性染色强度。 导出 每个门控总体的统计信息到Excel电子表格。

食谱

  1. 完成RPMI
    注意:在使用前,使用0.2μm过滤器进行灭菌。
    RPMI 500 ml
    50 ml热灭活的FBS
    5ml Pen-Strep-Glutamine 100x储液
  2. CyPBS
    用MilliQ水稀释10倍PBS至1倍,
  3. CyFACS缓冲区
    1x含0.1%BSA的CyPBS
    2mM EDTA和0.05%叠氮化钠 在MilliQ水中制造
    注意:不要使用FBS!
  4. 活死病
    5mg/ml装载有139-镧*或115-铟*的马来酰亚胺-DOTA *使用天然丰富的金属氯化物盐; > 95%规定同位素; 痕量金属纯99.99%
    将41.5mg/ml的固体DOTA-马来酰亚胺溶解在含有25mM金属(III)盐的硝酸(100ml 3%v/v硝酸/10ml水)中酸化的L-缓冲液(Fluidigm)或MilliQ水中。 在室温下孵育15分钟,然后等分并在-20℃下冷冻 41.5mg/ml对于DOTA马来酰亚胺为50mM。 DOTA:金属的2:1摩尔比旨在减少/消除条形码试剂中的未螯合金属。
    注意:每一批新的金属负载的马来酰亚胺-DOTA应该滴定,以最佳分辨率活对死细胞,具有最小的背景。因此,最终工作浓度和最终稀释度可能因批次而略有不同。

致谢

我们要感谢NIH拨款S10RR027582,5U19AI057229和5U19AI090019的资金。我们还要感谢Evan Newell博士的初始协议开发(Newell等人,2012年)。

参考文献

  1. Maecker,H. T.,McCoy,J. P.和Nussenblatt,R。(2012)。 人免疫学项目的免疫表型标准化。 Nat Rev Immunol 12(3):191-200。
  2. Newell,E.W.,Sigal,N.,Bendall,S.C.,Nolan,G.P.and Davis,M.M。(2012)。 按飞行时间的细胞计数显示组织细胞因子表达和连续区内的病毒特异性细胞壁 CD8 + T细胞表型。免疫 36(1):142-152。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Leipold, M. D. and Maecker, H. (2015). Phenotyping of Live Human PBMC using CyTOFTM Mass Cytometry. Bio-protocol 5(2): e1382. DOI: 10.21769/BioProtoc.1382.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。

Holden Maecker
Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, USA
While we haven't made a direct comparison, it should be fine to use FBS rather than BSA. Regards,

Holden
5/6/2018 4:58:35 PM Reply
tianli Tian
university of pennsylvania
Hi, May I ask why not use FBS for CyFACS buffer? have to use BSA?

Thanks!

Lifeng
5/6/2018 10:51:17 AM Reply