Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry

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The Human Immune Monitoring Center (HIMC)
0 2014



The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry or CyTOF, platform allows for the parallel application of about 40 labeled antibodies to a single sample, creating the possibility to read out many cell types and signaling pathways in a single small blood sample. We developed such a mass cytometry panel, consisting of 22 antibodies to cell surface lineage markers and 8 antibodies to phospho-specific epitopes of signaling proteins. These antibodies were chosen to discriminate all major white blood cell lineages, to a level of detail that includes subsets such as naïve, central memory, effector memory, and late effector CD4+ and CD8+T cells, naïve, transitional, and switched memory B cells, plasmablasts, myeloid and plasmacytoid dendritic cells, CD16+ and CD16+CD56+ NK cells, CD16+ and classical monocytes etc. 32 such cell subsets are defined in our standard gating scheme. The eight phospho-specific antibodies were chosen to represent major signaling nodes responsive to cytokine, TLR, and antigen receptor signaling. This antibody panel is used with 8 standard stimulation conditions (unstimulated, IFNa, IL-6, IL-7, IL-10, IL-21, LPS, PMA+ ionomycin), although other stimuli can be added. Comparison of healthy controls to subjects with immune deficiencies of unknown etiology may help elucidate the mechanisms of such deficiencies.

Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways, such as T and B cell signaling, those regulating apoptosis, growth and cell cycle control, plus those involved with cytokine, chemokine, and stress responses. Phosphoflow assays combine phospho-specific antibodies with the power of flow cytometry to enhance phospho protein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (Brand Name) metal-chelating polymers and permeabilized with methanol. They are then stained with intracellular phospho-specific antibodies.

We use a CyTOFTM mass cytometer to acquire the ICP-MS data. The current mass window selected is approximately AW 103-203, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. 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. Median values are reported to quantitate the level of phosphorylation of each protein in response to stimulation. Comparing the level of phosphorylation between samples can offer insight to the status of the immune system. Whole blood stimulation is the closest to the in vivo condition and it allows for assessment of granulocyte population as well as lymphocytes and monocytes.

Materials and Reagents

  1. Whole blood from patient or donor
  2. Cytokine aliquots (IFNα, IL-6, IL-7, IL-10, IL-21, LPS, PMA/Ionomycin etc.)
    1. IFNa (PBL Interferon source, catalog number: 11105-1 )
    2. IFNg2 (BD Biosciences, catalog number: 554617 )
    3. IL6 (BD Biosciences, catalog number: 550071 )
    4. IL7 (BD Biosciences, catalog number: 554608 )
    5. IL10 (BD Biosciences, catalog number: 554611 )
    6. IL21 (Life Technologies, Gibco®, catalog number: PHC0214 )
    7. IL2 (BD Biosciences, catalog number: 554603 )
    8. CD3 (BD Biosciences, catalog number: 555329 )
    9. CD28 (BD Biosciences, catalog number: 555725 )
    10. LPS (Sigma-Aldrich, catalog number: L7770 )
    11. IL5 (Pepro Tech, catalog number: 200-05 )
    12. IL17A (Pepro Tech, catalog number: 200-17 )
    13. IL17E (Pepro Tech, catalog number: 200-24 )
  3. Methanol (Thermo Fisher Scientific, catalog number: A452SK-1 )
  4. Dulbecco’s Phosphate-buffered saline (Ca2+, Mg2+)
  5. Phenotyping and phosphoprotein antibodies filtered with 0.1 um spin filters to get even staining of markers
  6. Ir-intercalator stock solution from Fluidigm Sciences (Rh103-intercalator can be used)
  7. 10x phosphate-buffered saline (Rockland, catalog number: MB-008 )
  8. Smart tube 1x thaw-lyse buffer (Smart Tube Inc.)
  9. Complete RPMI (see Recipes)
  10. CyFACS buffer (see Recipes)


  1. Nunc Coded Cryobank Vials (Cluster tubes, catalog number: 374078 )
  2. 37 °C water bath
  3. Biosafety cabinet
  4. Centrifuge
  5. CO2 incubator at 37 °C
  6. Calibrated pipettes
  7. 8 or 12 pin aspirator (V&P Scientific, model: Inc VP187A )
  8. Smart tube proteomic stabilizer (Smart Tube Inc.)


  1. Prepare stimulations in cluster tubes
    1. Prepare cytokines at 5x concentrations in Complete RPMI, with enough volume to pipette 50 µl into a well for each sample and control. See chart below for dilution for a full plate.
    2. The Cluster tubes with the aliquoted stimulants can be frozen away at -80 °C until further use.

  2. Example of a full plate

  3. Example of cytokine stimulations
    IFNα: Final concentration of stimulation used=10,000 units/ ml
    IFNγ2: Final concentration of stimulation used= 50 ng/ ml
    IL6: Final concentration of stimulation used= 50 ng/ ml
    IL7: Final concentration of stimulation used= 50 ng/ ml
    IL10: Final concentration of stimulation used= 50 ng/ ml
    IL21: Final concentration of stimulation used= 50 ng/ ml
    IL2: Final concentration of stimulation used= 50 ng/ ml
    CD3 = 2.5 μl in 990 ul (Final concentration 500 ng/ml)
    CD28 = 10 μl in above media (Final concentration 2,000 ng/ml)
    LPS: Final concentration of stimulation used= 1 μg/ ml
    PMA: 10 ng/ml final concentration /ml
    Ionomycin: 1,000 ng/ml final concentration /ml
    IL5: Final concentration of stimulation used= 10 ng/ ml
    IL17A: Final concentration of stimulation used= 50 ng/ ml
    IL17E: Final concentration of stimulation used= 50 ng/ ml

  4. Stimulation
    1. Rest the blood collected from donors in incubator at 37 °C in CO2 incubator for 1 h. Just before use, take out the required number of cluster tubes with the stimulant from the -80 °C freezer and let warm at 37 °C water bath for 5-10 min.
    2. Aliquot 200 μl of whole blood into column 1 of the cluster tube plate using a multichannel pipette. Change tips between each patient.
    3. Repeat with all the columns of tubes depending on the number of donors.
    4. Work as rapidly as possible.
    5. Tap plate to mix, and incubate 15 min at 37 °C in CO2 incubator.
    6. Remove stimulated whole blood from incubator at 15 min and using a multichannel pipette, add 250 µl of 1x Proteomic Stabilizer to each column of patient samples in the Cluster tube block. Pipette up and down to mix for each patient. Change tips between patients. Add the stabilizer in the same order that you added the whole blood for stimulation.
    7. Incubate for 10 min at room temperature. At this point it can be frozen away at -80 °C until it is ready to be Thaw-lysed and surface stained.

      Figure 1. A flow chart of the steps described in our procedure

  5. Surface staining
    1. Take out the required number of frozen stimulated cluster tubes with whole blood as can be comfortably run on the CyTOF in 1 day. (You can run 3-4 cluster or strip of samples in one day by CyTOF. One cluster or strip has 8 wells). Thaw in cold water for about 15 min.
    2. Using a pipette, transfer the fixed whole blood sample (450 μl) into a labeled deep well plate with 1.2 ml of 1x Thaw-Lyse and let it sit for 10 min at room temperature.
    3. Centrifuge cells at 548 rcf (x g) for 10 min at room temperature.
    4. Aspirate supernatant from the cells.
    5. Vortex the pellet and add 1.6 ml, 1x Thaw-Lyse, and let it sit for 5-10 min at room temperature. Centrifuge cells at 548 rcf (x g) for 10 min at room temperature.
    6. Aspirate supernatant from the cells.
    7. Vortex and wash the pellet with ~1.8 ml CyFACS.
    8. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C.
    9. Aspirate supernatant from the cells so that about 50 ul remains at the bottom of each well.
    10. Make cocktail in phosphate buffered saline of metal-chelating polymer-labeled surface antibodies according to previously determined titration. Make sufficient volume for each well to have 20 μl of cocktail. Pipet into 0.1 μm spin filter and centrifuge in a tabletop microcentrifuge (RCF=14,000) for 2 min at room temperature. This ensures even staining.
    11. Add 20 μl of antibody cocktail to the cells in the deep well plate, vortex to mix and let it incubate at room temperature for 30 min.
    12. Wash cells with 1.8 ml phosphate buffered saline and centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Aspirate.
    13. Permeabilize the cells by adding 600 µl cold methanol to each well of the deep well block using a multichannel pipette. Pipette up and down to mix for each patient. Change tips between patients. Cells are stored overnight at this point at -80 °C. This is done for convenience of work flow.
    14. Remove samples from freezer. Add 1.0 ml of CyFACS. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Aspirate so that about 100 μl remains in the wells.
    15. Wash in 1.8 ml/ well CyFACS buffer.
    16. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Discard supernatant by aspiration.

  6. Intracellular staining
    1. Make cocktail in PBS of metal-chelating polymer-labeled intracellular antibodies according to previously determined titration. Make sufficient volume for each sample to have 20 μl of cocktail. Pipet into 0.1 μm spin filter and centrifuge in tabletop microcentrifuge (RCF=14,000) for 2 min at room temperature.
    2. Add 20 μl of antibody cocktail to the cells in the deep well plate, vortex to mix and let it incubate at room temperature for 30 min.
    3. Add 1.6 ml of PBS. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Discard supernatant by aspiration.
    4. Make 1:200 dilution in PBS of Ir-intercalator. Add 20 μl of diluted Ir-intercalator solution to each sample, pipet to mix. Incubate on ice for 20 min.
    5. Wash 3 times in MilliQ water. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Discard supernatant by aspiration.
    6. Bring up the samples in 1 ml of MilliQ water and filter samples through a cell strainer. Acquire samples on the Cytof, after standard instrument setup procedures.

Representative data

Table 1. Antibodies in the phospho-CyTOF panel

Whole Blood Gating Scheme

Figure 2. Whole blood gating scheme


  1. Staining volumes are kept at a total of 50 to 60 μl.
  2. Washing steps are critical to minimize background. Aspirate such that only ~ 100 μl volume is left after each wash. Vortex before the next wash.
  3. Homogeneous staining is aided by thorough resuspension via pipetting up and down.
  4. Sample dilution is critical to collecting an optimal number of single cell events; aim for about 300 total events/sec.
  5. Do not leave sample buffer or wash buffers exposed to air, as dust accumulation can cause clogs.
  6. Each sample should be resuspended in water and filtered through nylon mesh just prior to running; do not let samples sit on water, as cells will lyse and clump, causing clogs and loss of events.


  1. Complete RPMI (Hyclone RPMI-1640 Medium)
    RPMI with 10% FBS
  2. CyFACS buffer
    1x CyPBS PBS with 0.1% BSA, and 0.05% Na azide
    Made in MilliQ water
    Note: Do not use FBS!


This work was supported by grants S10RR027582, 5U19AI057229, and 5U19AI090019 from the U.S. National Institutes of Health.


  1. Bendall, S. C., Simonds, E. F., Qiu, P., Amir el, A. D., Krutzik, P. O., Finck, R., Bruggner, R. V., Melamed, R., Trejo, A., Ornatsky, O. I., Balderas, R. S., Plevritis, S. K., Sachs, K., Pe'er, D., Tanner, S. D. and Nolan, G. P. (2011). Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332(6030): 687-696.


在一系列免疫细胞中评估一系列细胞因子,抗原受体和Toll样受体(TLR)信号通路的功能的能力可以提供人免疫系统状态的一种指纹。质谱仪或CyTOF平台允许将约40种标记的抗体平行应用于单个样品,产生在单个小血液样品中读出许多细胞类型和信号传导途径的可能性。我们开发了这样的大规模细胞仪板,由22个抗体的细胞表面沿袭标记和8抗体的磷酸特异性表位的信号蛋白。选择这些抗体以区分所有主要白细胞谱系,其细节水平包括诸如初始,中枢记忆,效应记忆和晚期效应CD4 +和CD8 + T细胞,天然,过渡和转换记忆B细胞的子集,浆母细胞,骨髓和浆细胞样树突状细胞,CD16 +和CD16 + CD56 + NK细胞,CD16 +和经典单核细胞等。在我们的标准门控方案中定义这样的细胞亚群。选择8种磷酸特异性抗体以代表响应细胞因子,TLR和抗原受体信号传导的主要信号转导节点。该抗体组与8种标准刺激条件(未刺激,IFNa,IL-6,IL-7,IL-10,IL-21,LPS,PMA +离子霉素)一起使用,尽管可以加入其它刺激。健康对照与具有未知病因的免疫缺陷的受试者的比较可能有助于阐明这种缺陷的机制。
酪氨酸,丝氨酸和苏氨酸残基的磷酸化对于控制参与各种细胞事件的蛋白质活性是至关重要的。激酶和磷酸酶的分类调节许多不同的细胞信号传导途径,例如T和B细胞信号传导,调节细胞凋亡,生长和细胞周期控制,加上那些参与细胞因子,趋化因子和应激反应的细胞内蛋白磷酸化。 Phosphoflow测定组合磷酸特异性抗体与流式细胞术的力量,以增强磷蛋白研究。在我们的测定中,外周血单核细胞被细胞因子刺激,固定,用标记有MAXPAR(Brand Name)金属螯合聚合物并用甲醇透化的抗体的混合物表面染色。然后用细胞内磷酸特异性抗体染色。
我们使用CyTOF TM 质谱仪获取ICP-MS数据。选择的当前质量窗口大约是AW 103-203,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。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。报道中值以定量响应刺激的每种蛋白质的磷酸化水平。比较样品之间的磷酸化水平可以提供对免疫系统状态的了解。全血刺激最接近体内条件,它允许评估粒细胞群以及淋巴细胞和单核细胞。


  1. 患者或捐赠者的全血
  2. 细胞因子等分试样(IFNα,IL-6,IL-7,IL-10,IL-21,LPS,PMA/Ionomycin等)
    1. IFNa(PBL干扰素来源,目录号:11105-1)
    2. IFNg2(BD Biosciences,目录号:554617)
    3. IL6(BD Biosciences,目录号:550071)
    4. IL7(BD Biosciences,目录号:554608)
    5. IL10(BD Biosciences,目录号:554611)
    6. IL21(Life Technologies,Gibco ,目录号:PHC0214)
    7. IL2(BD Biosciences,目录号:554603)
    8. CD3(BD Biosciences,目录号:555329)
    9. CD28(BD Biosciences,目录号:555725)
    10. LPS(Sigma-Aldrich,目录号:L7770)
    11. IL5(Pepro Tech,目录号:200-05)
    12. IL17A(Pepro Tech,目录号:200-17)
    13. IL17E(Pepro Tech,目录号:200-24)
  3. 甲醇(Thermo Fisher Scientific,目录号:A452SK-1)
  4. Dulbecco's磷酸盐缓冲盐水(Ca 2+,Mg 2+,Mg 2+)</sup>
  5. 用0.1μm旋转过滤器过滤表型和磷蛋白抗体,以获得标记物的均匀染色
  6. 来自Fluidigm Sciences的Ir-嵌入剂储备溶液(可以使用Rh103-嵌入剂)
  7. 10x磷酸盐缓冲盐水(Rockland,目录号:MB-008)
  8. 智能管1x解冻缓冲液(Smart Tube Inc.)
  9. 完成RPMI(参见配方)
  10. CyFACS缓冲区(参见配方)


  1. Nunc Coded Cryobank Vials(Cluster tubes,目录号:374078)
  2. 37°C水浴
  3. 生物安全柜
  4. 离心机
  5. CO 2培养箱中37℃培养
  6. 校准移液器
  7. 8或12针吸气器(V& P Scientific,型号:Inc VP187A)
  8. 智能管蛋白质稳定剂(Smart Tube Inc.)


  1. 准备簇管中的刺激
    1. 准备细胞因子在5x浓度在完全RPMI,足够 体积吸取50μl到每个样品和对照的孔中。 看到 下图为全板稀释的图表
    2. 具有等分的兴奋剂的簇管可以在-80℃下冷冻,直到进一步使用

  2. 满板示例

  3. 细胞因子刺激的实例
    IFNα:使用的刺激的最终浓度= 10,000单位/ml
    IFNγ2:使用的刺激的最终浓度= 50ng/ml
    IL6:使用的刺激的最终浓度= 50ng/ml
    IL7:使用的刺激的最终浓度= 50ng/ml
    IL10:所用刺激的最终浓度= 50ng/ml
    IL21:使用的刺激的最终浓度= 50ng/ml
    IL2:使用的刺激的最终浓度= 50ng/ml
    CD3 =2.5μl,在990μl(终浓度500ng/ml)中 CD28 =10μl在上述培养基(终浓度2,000ng/ml)中 LPS:使用的刺激的最终浓度=1μg/ml
    IL5:使用的刺激的最终浓度= 10ng/ml
    IL17A:使用的刺激的最终浓度= 50ng/ml
    IL17E:使用的刺激的最终浓度= 50ng/ml

  4. 刺激
    1. 将从供体收集的血液在37℃下在CO 2培养箱中在培养箱中休息1小时。 在使用前,取出所需的数量 簇管用来自-80℃冰箱的兴奋剂并让其温热   37℃水浴5-10分钟。
    2. 等分200μl全血 使用多通道移液管进入簇管板的柱1。 更改每个病人之间的提示。
    3. 根据捐赠者的数量重复所有的管柱。
    4. 工作尽可能快。
    5. 敲击板混合,并在37℃下在CO 2孵育器中孵育15分钟。
    6. 从培养箱中取出受刺激的全血15分钟,并使用 多通道移液器,每个添加250微升1蛋白质组稳定剂 列的患者样品在群集管块。 移液器上下移动   以针对每个患者进行混合。 更改患者之间的提示。 加上 稳定剂以你添加全血的顺序 刺激
    7. 在室温下孵育10分钟。 在这 点,其可以在-80℃冷冻,直到其准备解冻   并表面染色

      图1。 我们的程序中所述步骤的流程图

  5. 表面染色
    1. 取出所需数量的冷冻刺激簇管 全血可以舒适地运行在CyTOF在1天。 (您可以 通过CyTOF在一天内运行3-4簇样品或条带。 一个集群或 条有8口井)。 在冷水中解冻约15分钟。
    2. 使用a 移液管,将固定的全血样品(450μl)转移到标记的 深孔板用1.2ml的1x Thaw-Lyse,并让它坐10分钟   室温。
    3. 在548rcf(×g/g)离心细胞10分钟,在室温。
    4. 吸出细胞的上清液。
    5. 涡旋球团,并加入1.6毫升,1解冻溶胞,让它坐 在室温下5-10分钟。 离心细胞在548 rcf( x g )10 min。
    6. 吸出细胞的上清液。
    7. 涡旋并用约1.8ml CyFACS洗涤沉淀
    8. 在4℃下以974rcf(×g/g)离心细胞10分钟。
    9. 吸出来自细胞的上清液,使得约50ul保留在每个孔的底部。
    10. 在磷酸盐缓冲盐水的金属螯合制备鸡尾酒 聚合物标记的表面抗体 滴定。 使每个孔有足够的体积有20微升 鸡尾酒。 吸入0.1微米旋转过滤器和离心机在桌面 微量离心机(RCF = 14,000)在室温下2分钟。 这确保   甚至染色。
    11. 加入20微升的抗体鸡尾酒的细胞 深孔板,涡旋混合并让其在室温下孵育 温度30分钟。
    12. 用1.8ml磷酸盐缓冲盐水洗涤细胞,并在4℃下以974rcf(×g/g)离心细胞10分钟。 吸气。
    13. 通过向每个孔中加入600μl冷甲醇来透化细胞 的深井块使用多通道移液器。 移液器上下移动   以针对每个患者进行混合。 更改患者之间的提示。 储存细胞   在该点过夜,在-80℃。 这是为了方便工作 流。
    14. 从冷冻机中取出样品。 加入1.0ml CyFACS。 在4℃下以974rcf(×g/g)离心细胞10分钟。 吸出这样 约100μl保留在孔中。
    15. 以1.8ml /孔的CyFACS缓冲液洗涤
    16. 在4℃下以974rcf(×g/g)离心细胞10分钟。 通过抽吸弃去上清液。

  6. 细胞内染色
    1. 在PBS的金属螯合聚合物标记细胞内的鸡尾酒 抗体。 充足   每个样品的体积具有20μl的混合物。 移入0.1μm 旋转过滤和离心机在台式微量离心机(RCF = 14000)2   min。
    2. 加入20μl的抗体鸡尾酒 细胞在深孔板中,涡旋混合并让其在室温下孵育 温度30分钟
    3. 加入1.6ml PBS。 在4℃下以974rcf(×g/g)离心细胞10分钟。 抽吸弃去上清液。
    4. 在Ir-嵌入剂的PBS中进行1:200稀释。 加入20μl稀释 Ir-嵌入剂溶液加到每个样品中,用移液管混合。 在冰上孵化 20分钟。
    5. 在MilliQ水中洗涤3次。 在4℃下以974rcf(×g/g)离心细胞10分钟。 抽吸弃去上清液。
    6. 将样品置于1ml MilliQ水中,过滤样品 通过细胞过滤器。 标准后获得Cytof样品 仪器设置程序。


表1. 磷酸化-CyTOF平板中的抗体




  1. 染色体积保持在总共50至60μl。
  2. 洗涤步骤是最小化背景的关键。 吸出使每次洗涤后仅剩约〜100μl体积。 在下一次洗涤之前涡旋
  3. 通过上下移液彻底重悬,有助于均匀染色
  4. 样品稀释对于收集最佳数量的单细胞事件至关重要; 目标为约300次事件/秒。
  5. 不要将样品缓冲液或洗涤缓冲液暴露在空气中,因为灰尘积累会导致堵塞
  6. 每个样品应该重新悬浮在水中,并在运行之前通过尼龙网过滤; 不要让样品坐在水面上,因为细胞会溶解和结块,导致堵塞和事件的丢失。


  1. 完全RPMI(Hyclone RPMI-1640 Medium)
  2. CyFACS缓冲区
    1x含有0.1%BSA的CyPBS PBS和0.05%叠氮化钠 在MilliQ水中制造




  1. Bendall,SC,Simonds,EF,Qiu,P.,Amir el,AD,Krutzik,PO,Finck,R.,Bruggner,RV,Melamed,R.,Trejo,A.,Ornatsky,OI,Balderas,RS,Plevritis ,SK,Sachs,K.,Pe'er,D.,Tanner,SD和Nolan,GP(2011)。 跨越人类造血连续区的不同免疫和药物反应的单细胞质谱仪。 332(6030):687-696。
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引用:Fernandez, R. and Maecker, H. (2015). Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry. Bio-protocol 5(11): e1495. DOI: 10.21769/BioProtoc.1495.