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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. These pathways include T and B cell signaling, regulating growth and cell cycle control, plus cytokine, chemokine, and stress responses. Phosphoflow assays combine phosphoprotein-specific antibodies with the power of flow cytometry to enhance phosphoprotein 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 (inductively coupled plasma mass spectrometry) 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.

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Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry
CyTOF质谱流式细胞仪测定外周血单核细胞中细胞因子刺激后的磷酸化变化

免疫学 > 免疫细胞染色 > 免疫检测
作者: Rosemary Fernandez
Rosemary FernandezAffiliation: Human Immune Monitoring Center, Stanford University, Stanford, USA
Bio-protocol author page: a2282
 and Holden Maecker
Holden MaeckerAffiliation: Human Immune Monitoring Center, Stanford University , Stanford, USA
For correspondence: maecker@stanford.edu
Bio-protocol author page: a1861
Vol 5, Iss 11, 6/5/2015, 3908 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1496

[Abstract] 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. These pathways include T and B cell signaling, regulating growth and cell cycle control, plus cytokine, chemokine, and stress responses. Phosphoflow assays combine phosphoprotein-specific antibodies with the power of flow cytometry to enhance phosphoprotein 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 (inductively coupled plasma mass spectrometry) 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.

[Abstract]

Materials and Reagents

  1. PBMC (fresh or thawed frozen)
  2. Benzonase (Pierce Antibodies, catalog number: 88701 )
  3. Cytokine aliquots (IFNα, IFNγ, IL-6, IL-7, IL-10, IL-21, IL-2 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 )
  4. 16% PFA (Alfa Aesar, catalog number: 4368 )
  5. Methanol (Thermo Fisher Scientific, catalog number: A452SK-1 )
  6. Deep Well plate (Costar, catalog number: 3960 )
  7. Phenotyping and phosphoprotein antibodies filtered with 0.1 um spin filters (EMD Millipore, model: UFC30VV00 )
  8. Ir-intercalator stock solution from DVS (Rh103-intercalator can be used) (catalog number: 201192 B )
  9. 1x CyPBS PBS (Rockland, catalog number: MB-008 )
  10. Complete RPMI (see Recipes)
  11. CyFACS buffer (see Recipes)

Equipment

  1. 37 °C water bath
  2. Biosafety cabinet
  3. Centrifuge
  4. CO2 incubator at 37 °C
  5. Calibrated pipettes
  6. 8 or 12 pin aspirator (V&P Scientific, model: VP187A )

Procedure

  1. Thaw PBMC
    1. Warm complete RPMI to 37 °C in water bath. Each sample will require 20 ml of complete RPMI with benzonase to limit cell clumping. Calculate the amount needed to thaw all samples, and prepare a separate aliquot of warm media with 1:10,000 benzonase (final concentration 25 U/ml). Remove samples from liquid nitrogen and transport to lab on dry ice.
    2. Place 10 ml of warmed benzonase media into a 15 ml tube, making a separate tube for each sample.
    3. Thaw frozen vials in 37 °C water bath.
    4. When cells are partially thawed, carry to hood.
    5. 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.
    6. Continue with the rest of the samples as quickly as possible.
    7. Centrifuge cells at 1,600 rpm (RCF = 390) for 10 min at room temperature.
    8. Remove supernatant from the cells and resuspend the pellet by tapping the tube.
    9. Gently resuspend the pellet in 1 ml warmed benzonase media. Centrifuge cells at 1,600 rpm (RCF = 390) for 10 min at room temperature. Remove supernatant from the cells and resuspend the pellet by tapping the tube.
    10. Resuspend cells in 1 ml warm complete RPMI.
    11. Count cells with Vicell (Vi-Cell XR Beckman Coulter) (or hemocytometer if necessary). To count, take 20 µl cells and dilute with 480 µl PBS in vicell counting chamber. Load onto Vicell as PBMC with a 1:25 dilution factor.
    12. Adjust the cell concentration to 5 x 106 cells/ml with warm media (no more benzonase at this point.)
    13. Using a multichannel pipette, add 100 µl cells (0.5 x 106 cells) into each of eight wells of a 96-well deep well plate.
    14. Rest for another 1 h-1.5 h at 37 °C in CO2 incubator. Prepare the stimulation plate just before stimulation.
      Example of a full plate:


  2. Stimulate cells
    1. Prepare cytokines at 5x concentrations, in 500 µl of RPMI in a fresh deep well block.
    2. Add enough 5x cytokine (~500 µl for a full plate) into one row of a fresh deep well block to pipette.
      Example of cytokine stimulation:
      IFNa: Final concentration of stimulation used= 10,000 units/ml
      IFNg2, IL6, IL7, IL10, IL21, IL2, IL17A: Final concentration of stimulation used= 50 ng/ ml
      CD3 = 2.5 µl in 990 µl (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
    3. Remove rested cells in the deep well block from incubator and stimulate by adding 25 µl of 5x cytokines with multichannel pipette to each row of patient samples. Change tips between each patient. Work as rapidly as possible.
    4. Tap plate to mix, and incubate 15 min at 37 °C in CO2 incubator.
    5. Remove cells from incubator at the 15 min and using a multichannel pipette, add 20 µl 16% PFA (~2.2% PFA final concentration) to each row of patient samples in the deep well block. Pipette up and down to mix for each patient. Change tips between patients. Add PFA in the same order that you added the cytokine stimulation.
    6. Incubate 10 min at room temperature.
    7. Add 1.6 ml CyFACS buffer to each well of the deep well block.
    8. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. The cells are fixed / dead and has to be spun down harder to prevent cell loss.
    9. Aspirate supernatant from the cells.

  3. Surface staining
    1. Make cocktail in PBS of metal-chelating polymer-labeled surface 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 a tabletop microcentrifuge (RCF = 14,000) for 5 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 RT for 30 min.
    3. Wash cells in 1.6 ml/well x2 with CyFACS buffer and centrifuge cells at 974 rcf (x g) for 8 min at 4 °C. Discard supernatant by aspiration.
    4. Permeabilize the cells by adding 600 µl -20 °C cold MeOH 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.
    5. Cells are stored overnight at this point at -80 °C.
    6. Remove samples from freezer.
    7. Wash in 1 ml/well CyFACS buffer.
    8. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Discard supernatant by aspiration.
    9. Wash in 1.8 ml / well CyFACS buffer.
    10. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Discard supernatant by aspiration.

  4. 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 a tabletop microcentrifuge (RCF = 14,000) for 5 min at room temperature.
    2. Add 20 µl of antibody cocktail to the cells in the deep well plate and let it incubate at RT for 30 min.
    3. Wash in 1.8 ml/ well CyPBS.
    4. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Discard supernatant by aspiration.
    5. Make 1:200 dilution in CyPBS of Ir-intercalator. Add 20 µl of diluted Ir-intercalator solution to each well, pipet to mix. Incubate on ice for 20 min.
    6. Wash 1.6 ml/ well x 3 in MilliQ water.
    7. Centrifuge cells at 974 rcf (x g) for 10 min at 4 °C. Discard supernatant by aspiration.
    8. Acquire samples on the CyTOF, after standard instrument setup procedures.

Representative data

  1. Gating of Surface markers


    Figure 1. Gating of cell-surface markers in a healthy control PBMC sample


    Figure 2. Phospho-signaling as detected in CD4+ and CD8+ T cells with a variety of stimuli (US=unstimulated)

Notes

  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 / second.
  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 overnight, as cells will lyse and clump, causing clogs and loss of events.

Recipes

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

Acknowledgments

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

References

  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.

材料和试剂

  1. PBMC(新鲜或解冻冷冻)
  2. Benzonase(Pierce Antibodies,目录号:88701)
  3. 细胞因子等分试样(IFNα,IFNγ,IL-6,IL-7,IL-10,IL-21,IL-2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)
  4. 16%PFA(Alfa Aesar,目录号:4368)
  5. 甲醇(Thermo Fisher Scientific,目录号:A452SK-1)
  6. 深井板(Costar,目录号:3960)
  7. 用0.1μm旋转过滤器(EMD Millipore,型号:UFC30VV00)过滤的表型和磷蛋白抗体
  8. 来自DVS的Ir-嵌入剂储备溶液(可以使用Rh103-嵌入剂)(目录号:201192B)
  9. 1×CyPBS PBS(Rockland,目录号:MB-008)
  10. 完成RPMI(参见配方)
  11. CyFACS缓冲区(参见配方)

设备

  1. 37°C水浴
  2. 生物安全柜
  3. 离心机
  4. CO 2培养箱中37℃培养
  5. 校准移液器
  6. 8或12针吸气器(V& P Scientific,型号:VP187A)

程序

  1. 解冻PBMC
    1. 温热完成RPMI至37°C水浴。 每个样品将需要20 ml的完全RPMI与benzonase以限制细胞聚集。 计算   解冻所有样品所需的量,并制备单独的等分试样 温热培养基与1:10,000 benzonase(终浓度25U/ml)。 去掉   来自液氮的样品并在干冰上运输到实验室
    2. 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。
    3. 在37℃水浴中解冻冷冻的小瓶。
    4. 当细胞被部分解冻,携带到罩。
    5. 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。
    6. 尽快继续其余样品。
    7. 在室温下以1,600rpm(RCF = 390)离心细胞10分钟
    8. 从细胞中除去上清液,并通过轻敲管子重悬沉淀
    9. 轻轻地将沉淀重悬在1ml温热的benzonase培养基中。 在室温下以1,600rpm(RCF = 390)离心细胞10分钟 温度。 从细胞中取出上清液并重悬沉淀 通过点击管
    10. 将细胞重悬于1ml温热完全RPMI中
    11. 用Vicell(Vi-Cell XR Beckman Coulter)(或 如果必要,血细胞计数器)。 计数,取20微升细胞并用稀释 480μlPBS在vicell计数室中。 作为PBMC加载到Vicell上 1:25稀释因子。
    12. 用温热培养基(此时不再有benzonase)将细胞浓度调节至5×10 6个细胞/ml。
    13. 使用多通道移液器,向96孔深孔板的8个孔的每个孔中加入100μl细胞(0.5×10 6个细胞)。
    14. 在CO 2培养箱中于37℃静置另外1小时-1.5小时。 在刺激之前准备刺激板。
      全板示例:


  2. 刺激细胞
    1. 在新鲜的深井块中,在500μlRPMI中制备5x浓度的细胞因子。
    2. 添加足够的5x细胞因子(〜500微升全板)到一排新鲜的深井块移液器。
      细胞因子刺激的实例:
      IFNa:使用的刺激的最终浓度= 10,000单位/ml
      IFNg2,IL6,IL7,IL10,IL21,IL2,IL17A:使用的刺激的最终浓度= 50ng/ml
      CD3 =2.5μl,在990μl(终浓度500ng/ml)中 在上述培养基终浓度(浓度2,000ng/ml)中CD28 =10μl LPS:使用的刺激的最终浓度=1μg/ml
      PMA 10ng/ml终浓度ml
      离子霉素1000ng/ml终浓度/ml
      IL5:使用的刺激的最终浓度= 10ng/ml
    3. 从孵化器中取出深井块中的休眠细胞 通过加入25μl的5x细胞因子用多通道移液器刺激 每行患者样品。 更改每个病人之间的提示。 工作作为 尽快。
    4. 敲击板混合,并在37℃下在CO 2孵育器中孵育15分钟。
    5. 删除细胞从孵化器在15分钟,并使用多通道 移液管,加入20μl16%PFA(〜2.2%PFA终浓度)到每一行 的深井块中的患者样品。 吸移管向上和向下混合 。 更改患者之间的提示。 在同一个添加PFA 您添加细胞因子刺激的顺序
    6. 在室温下孵育10分钟。
    7. 向深井块的每个孔中加入1.6ml CyFACS缓冲液。
    8. 在4℃下以974rcf(×g/g)离心细胞10分钟。 细胞是 固定/死亡,必须更难旋转以防止细胞丢失
    9. 吸出细胞的上清液。

  3. 表面染色
    1. 在金属螯合聚合物标记的表面的PBS中制备鸡尾酒 抗体。 充足   每个样品的体积具有20μl的混合物。 移入0.1μm 旋转过滤器并在台式微量离心机中离心(RCF = 14,000) 在室温下搅拌5分钟
    2. 加入20μl的抗体混合物到深孔板中的细胞,涡旋混合,并让其在室温下孵育30分钟。
    3. 用CyFACS缓冲液和离心细胞以1.6ml /孔x2洗涤细胞   在974rcf(em x g)在4℃下8分钟。 通过抽吸弃去上清液。
    4. 通过加入600微升-20°C冷MeOH通过细胞 孔的深井块使用多通道移液器。 吸取和   下来混合为每个病人。 更改患者之间的提示。
    5. 细胞在这一点在-80℃下储存过夜
    6. 从冷冻机中取出样品。
    7. 以1ml /孔CyFACS缓冲液洗涤
    8. 在4℃下以974rcf(×g/g)离心细胞10分钟。 抽吸弃去上清液。
    9. 以1.8ml /孔的CyFACS缓冲液洗涤
    10. 在4℃下以974rcf(×g/g)离心细胞10分钟。 通过抽吸弃去上清液。

  4. 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。 使 每个样品有足够的体积具有20μl的鸡尾酒。 吸入 0.1μm旋转过滤器并在台式微量离心机(RCF = 14,000)在室温下处理5分钟
    2. 加入20μl的抗体混合物到深孔板中的细胞,并让其在室温孵育30分钟。
    3. 以1.8ml /孔CyPBS洗涤
    4. 在4℃下以974rcf(×g/g)离心细胞10分钟。 抽吸弃去上清液。
    5. 在Ir-嵌入剂的CyPBS中进行1:200稀释。 加入20μl 稀释的Ir-嵌入剂溶液到每个孔中,用移液管混合。 孵化   冰20分钟。
    6. 在MilliQ水中洗涤1.6ml /孔x 3。
    7. 在4℃下以974rcf(×g/g)离心细胞10分钟。 抽吸弃去上清液。
    8. 在标准仪器设置程序后,在CyTOF上采集样品。

代表数据

  1. 表面标记的选通


    图1。 健康对照PBMC样品中的细胞表面标记的门控


    图2. 在具有多种刺激(US =未刺激)的CD4 + 和CD8 + T细胞中检测到的磷酸信号

笔记

  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; 旨在约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; 不要让样品在水中过夜,因为细胞会溶解和结块,导致堵塞和事件的丢失。

食谱

  1. 完全RPMI(Hyclone RPMI-1640 Medium)
    含10%FBS的RPMI
    青霉素/链霉素
    谷氨酰胺
  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
    注意:不要使用FBS!

致谢

这项工作得到来自美国国立卫生研究院的拨款S10RR027582,5U19AI057229和5U19AI090019的支持。

参考文献

  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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How to cite this protocol: Fernandez, R. and Maecker, H. (2015). Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry. Bio-protocol 5(11): e1496. DOI: 10.21769/BioProtoc.1496; Full Text



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9/7/2016 5:44:08 AM  

Yvonne Vercoulen
UMCU

Hi, for phospho-flow analysis we are trying to induce clear T cell receptor signaling in total isolated PBMC (thawed). In Jurkat and other cell lines, as well as mouse T cells we can do it, but in total human PBMC we are not successful when utilizing aCD3/CD28 beads, or aCD3 antibody stimulation, and using P-ERK, P-S6 or P-Zap70 as readout. I noticed that you see some nice signaling profiles for cytokine stimulation. Does aCD3 or CD28 stimulation work for you on total PBMC? If so, can you please recommend a method to us?
Thank you & best wishes,
Yvonne Vercoulen

10/31/2016 8:42:07 AM  

Holden Maecker (Author)
Human Immune Monitoring Center, Stanford University , USA

Yes, we use CD3+CD28, either beads or soluble Ab, to stimulate PBMC for phospho-flow. For CD3+CD28 Dynabeads:
1. Exchange the supernatant of the Dynabeads with an equal volume of complete medium, by pelleting the beads with a magnetic separator.
2. Add 50 uL of Dynabeads in medium to 10^6 thawed, rested PBMC in a 96-well plate.
3. Stimulate for 30 min, then fix and proceed as per the published protocol.

10/31/2016 3:08:44 PM  

Rosemary Fernandez (Author)
Human Immune Monitoring Center, Stanford University, USA

When using soluble CD3, I stimulate for 15 minutes and fix.
CD3 = 2.5 ul in 990ul ( Final conc 500ng/ml) BD Pharmingin Catalog # 555329
CD28 = 10 ul in above media Final ( conc 2000ng / ml) BD Pharmingin Catalog #555725
I get very good stimulation of pP38, pPLCg2,pERK1/2, pS6, pSTAT3, IkB, pSTAT5 and
moderate stimulation of pAKT and pSTAT1

10/31/2016 11:08:13 PM  

Yvonne Vercoulen
UMCU

thanks, very helpful!

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