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Intracellular Cytokine Staining on PBMCs Using CyTOFTM Mass Cytometry
适用于CyTOF质谱流式细胞仪的外周血单核细胞(PBMC)细胞因子胞内染色方法   

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Abstract

In this protocol, we use a CyTOFTM mass cytometry to collect single-cell data on a large number of cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. The current selected mass window in AW 103-203 includes the lanthanides used for most antibody labeling, along with iridium and rhodium for DNA intercalators. The output data are in the format as .txt and .fcs files, which is compatible with many analysis programs. This protocol could be adapted to include tetramers into the staining panel, but we have not optimized for that purpose.
The principal steps of intracellular cytokine staining are as follows: First, cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail. An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the cytokines within the cell. Next, EDTA is added to remove adherent cells from the activation vessel. After washing, antibodies to cell surface markers are added to the cells. The cells are then fixed in paraformaldehyde and permeabilized. We use a gentle detergent, saponin, as the permealization buffer because it is less destructive to surface and intracellular epitopes compared to harsh detergents or methanol. After permeabilization, the metal-conjugated anti-cytokine antibodies are added into the cell suspension. The stained cells are then sequentially introduced into the mass cytometry for signal intensity analysis.

Materials and Reagents

  1. PBMC (fresh or thawed frozen)
  2. RPMI-1640 (Hyclone, catalog number: SH30027.01 )
  3. FBS (Atlanta Biologicals, catalog number: S11150 )
  4. Pen-strep-Glutamin 100X (Hyclone, catalog number: SV30082.01 )
  5. Benzonase (2.5 x 105 U/ml) (Pierce, catalog number: 88701 )
  6. Brefeldin A (Sigma-Aldrich, catalog number: B7651 )
  7. Monensin (Sigma-Aldrich, catalog number: M5273 )
  8. 0.5 M EDTA (Hoefer, catalog number: GR-123-100 )
  9. Sodium azide (10% w/v solution) (Teknova, catalog number: S0209 )
  10. 16% para-formaldehyde (PFA) (Alfa Aesar, catalog number: 43368 ))
  11. 10x PBS (ROCKLAND, catalog number: MB-008 )
  12. BSA (Sigma-Aldrich, catalog number: A7284 )
  13. Maleimide-DOTA (Macrocyclics, catalog number: B-272 )
  14. Lanthanum (III) chloride heptahydrate (Sigma-Aldrich, catalog number: 203521 )
  15. Indium (III) chloride (Sigma-Aldrich, catalog number: 203440 )
  16. MilliQ water
    Note: Beakers or bottles used here are not washed with soap due to barium content of most commercial soaps.
  17. Phenotyping antibodies (filtered with 0.1 µm spin filters) (Millipore, catalog number: UFC30VV00 )
  18. Ir-intercalator stock solution (Fluidigm, catalog number: 201192 )
    Note: Rh103-intercalator can be used.
  19. 10x saponin-based permeabilization buffer (eBioscience, catalog number: 00 8333-56 )
  20. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
  21. Ionomycine (Sigma-Aldrich, catalog number: I0634 )
  22. Phytohemagglutinin (PHA) (Sigma-Aldrich, catalog number: 61764 )
  23. SEB (Sigma-Aldrich, catalog number: S0812 )
  24. Anti-CD3/CD28 (various vendors)
  25. Peptide mixes (JPT)
  26. Complete RPMI (see Recipes)
  27. CyPBS (see Recipes)
  28. CyFACS buffer (see Recipes)
  29. Live-dead stain (see Recipes)

Equipment

  1. 96- well round-bottom plates
  2. 37 °C water bath
  3. Biosafety cabinet
  4. Centrifuge
  5. CO2 incubator at 37 °C
  6. Calibrated pipettes

Procedure

  1. Thaw PBMC
    1. Warm complete RPMI media to 37 °C in water bath. Each sample will require 22 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 (final concentration 25 U/ml). Benzonase is added into the media to prevent dead cell aggregation. Thaw no more than 3 samples at a time. Run one control PBMC with each batch of samples.
    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 1,550 rpm (RCF = 473) for 8 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. Add 9 ml more warmed benzonase media to the tube.
    11. Centrifuge cells at 1,550 rpm (RCF = 473) for 8 min at room temperature. Remove supernatant from the cells and resuspend the pellet by tapping the tube.
    12. Resuspend cells in 1 ml warm media.
    13. Count cells with Vicell (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.
    14. Adjust the cell concentration to 5-10 x 106 cells/ml with warm media (no more benzonase at this point).
    15. Using a multichannel pipette, add 200 µl cells (for at least 1 x 106 cells) into each well of a 96-well deep well plate. Split each sample equally into two or more wells keeping one as an unstimulated control and the others for different types of stimulation.
    16. Rest overnight (6-18 h) at 37 °C in CO2 incubator.

  2. Stimulate cells
    1. After overnight rest at 37 °C, add the activation reagents and secretion inhibitor (brefeldin A/monensin) to the well for stimulation. Add only the secretion inhibitor to the unstimulated control well. If doing CD107a staining, add CD107a antibody during the stimulation.

      Table 1. Protein secretion inhibitors
      Reagent
      Stock concentration
      Intermediate dilution
      Final concentration
      Brefeldin A
      5 mg/ml in DMSO
      (stored in aliquots at -20 °C)
      1:10 in PBS
      10 μg/ml (1:50) or 5 μg/ml (1:100) with monensin
      Monensin
      5 mg/ml in ethanol
      (stored at -20 °C)
      1:10 in PBS
      10 μg/ml (1:50) or 5 μg/ml (1:100) with brefeldinA

      Table 2. Activators
      Reagent
      Stock concentration
      Intermediate dilution
      Final concentration
      Phorbol 12-myristate 13-acetate (PMA)
      1 mg/ml in DMSO
      (stored in aliquots at -20 °C)
      1:1,000 in PBS
      10 ng/ml
      Ionomycine
      1 mg/ml in DMSO
      (stored in aliquots at -20 °C)
      1: 10 in PBS
      1 µg/ml
      Phytohemagglutinin (PHA)
      1 mg/ml in DMSO
      (stored at 4 °C)
      1:10 in PBS
      1 µg/ml
      SEB
      50 μg/ml in PBS
      None
      1 μg/ml (1:50)

      Anti-CD3/CD28
      Follow manufacturer instruction
      -
      -
      Peptide mixes
      0.5-1 mg/ml/pep in DMSO
      (stored in aliquots at -20 °C)
      1:10 in PBS
      1 μg/ml/peptide
      (1:50 - 1:100)

      Notes:
      1. It is important to avoid solvent toxicity. Final DMSO+ethanol concentration from all sources (peptides, brefeldin A, monensin) should not exceed 0.5%.
      2. For most cytokines: Use brefeldin A at 10 μg/ml final concentration (see stock preparation table). For CD107 and CD154: Use monensin at 10 μg/ml final concentration (see stock preparation table). For assays combining cytokines and CD107 or CD154: Use brefeldin A and monensin at 5 μg/ml final concentration each.
      3. Metal-labeled CD107 and CD154 can be added into the culture during the stimulation at a concentration of 2 μg/ml. This allows for staining of target molecules that are re-internalized by cells during the activation process.
      4. Addition of costimulatory antibodies is optional. Add 1 μg/ml final concentration of CD28 and/or CD49d (labeled antibody can be used if analysis of the marker is desired).
    2. Incubate the cells for 4 h (PMA + ionomycin stimulation, PHA + ionomycin stimulation) or 6-8 h (SEB, anti-CD3/CD28 stimulation, peptide stimulation) at 37 °C, in a CO2 incubator.
      Note: For most cytokines 6-12 h incubation at 37 °C is sufficient; For IL-10 optimal incubatation time is 12-24 h, but it is possible to detect in 6 h.
    3. At the end of stimulation, add EDTA to a final concentration of 2 mM and incubate for 15 min at room temperature.

  3. Staining
    1. Wash 2x with 250 µl CyFACS buffer per well 1,550 rpm (RCF = 473), 10 min at room temperature. The same volume and centrifuge conditions are used in additional wash steps before fixation with PFA (step C6).
    2. Make surface Ab cocktail in CyFACS buffer (filter with 0.1 µm spin filter) 100 µl per reaction. Incubate on ice for 45 min. Use vendor’s recommended concentration (or optimal titer as determined for self-made conjugates) for each antibody.
    3. Wash 2x in CyFACS buffer.
    4. Resuspend cells in 100 µl of 1:3,000 diluted 5 mg/ml 115-DOTA maleimide (titrated if new stock) in CyPBS, Incubate 30 min on ice.
    5. Wash 3x in CyFACS buffer.
    6. Resuspend in 100 µl of 2% PFA in CyPBS, Incubate 4 °C overnight.
    7. Wash 2x in 1x eBioscience perm buffer (1x in milliQ water), 2,000 rpm (RCF = 787), 10 min at 4 °C. The same volume and centrifuge conditions are used in the following wash steps.
    8. Make intracellular staining cocktail in 1x perm buffer and filter with 0.1 µm spin filter, 100 µl per reaction. Incubate on ice for 45 min.
    9. Wash 3x in CyFACS buffer.
    10. Resuspend in 100 µl 1: 2,000 Ir-Interchelator diluted in 2% PFA (in CyPBS).
    11. Incubate 20 min at room temp.
    12. Wash 2x in CyFACS buffer.
    13. Wash 3x in MilliQ water.
    14. Resuspend in MilliQ water (1 to 1.5 ml) for running on CyTOF.

Representative data



Figure 1. Flowjo gating of intracelluar markers upon PMA/Ionomycin stimulation

Recipes

  1. Complete RPMI
    RPMI1640
    10% FBS
    1x pen-strep -Glutamine
  2. CyPBS
    1x PBS without heavy metal contaminants, such as 10x PBS
    Made in MilliQ water
  3. CyFACS buffer
    1x CyPBS with 0.1% BSA
    2 mM EDTA and 0.05% sodium 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%)

Acknowledgments

This work was supported by grants # S10RR027582 and 5U19AI057229 from the National Institutes of Health. We thank Evan Newell and Mark Davis for help in the initial protocol development.

References

  1. 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.
  2. 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.
  3. Maecker, H. T. (2009). Multiparameter flow cytometry monitoring of T cell responses. Methods Mol Biol 485: 375-391.

简介

在这个协议中,我们使用CyTOF TM 质谱仪收集大量细胞因子/趋化因子以及表征T细胞和其他免疫细胞的细胞表面蛋白的单细胞数据。 AW 103-203中当前选择的质量窗口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。输出数据的格式为.txt和.fcs文件,与许多分析程序兼容。该方案可以适于将四聚体包括在染色板中,但是我们没有为此目的进行优化。细胞内细胞因子染色的主要步骤如下:首先,细胞被活化几小时,使用特异性肽或非特异性活化混合物。加入蛋白质转运抑制剂(例如布雷菲德菌素A)以将细胞因子保留在细胞内。接下来,加入EDTA以从活化容器中除去粘附细胞。洗涤后,将细胞表面标记物的抗体加入细胞中。然后将细胞固定在多聚甲醛中并透化。我们使用温和的洗涤剂皂苷作为渗透缓冲液,因为与苛刻的洗涤剂或甲醇相比,它对表面和细胞内表位的破坏性更小。透化后,将金属缀合的抗细胞因子抗体加入细胞悬浮液中。然后将染色的细胞依次导入质谱仪进行信号强度分析

材料和试剂

  1. PBMC(新鲜或解冻冷冻)
  2. RPMI-1640(Hyclone,目录号:SH30027.01)
  3. FBS(Atlanta Biologicals,目录号:S11150)
  4. Pen-strep-Glutamin 100X(Hyclone,目录号:SV30082.01)
  5. Benzonase(2.5×10 5 U/ml)(Pierce,目录号:88701)
  6. 布雷菲德菌素A(Sigma-Aldrich,目录号:B7651)
  7. 莫能菌素(Sigma-Aldrich,目录号:M5273)
  8. 0.5 M EDTA(Hoefer,目录号:GR-123-100)
  9. 叠氮化钠(10%w/v溶液)(Teknova,目录号:S0209)
  10. 16%多聚甲醛(PFA)(Alfa Aesar,目录号:43368))
  11. 10x PBS(ROCKLAND,目录号:MB-008)
  12. BSA(Sigma-Aldrich,目录号:A7284)
  13. 马来酰亚胺-DOTA(Macrocyclics,目录号:B-272)
  14. 氯化镧(III)七水合物(Sigma-Aldrich,目录号:203521)
  15. 氯化铟(III)(Sigma-Aldrich,目录号:203440)
  16. MilliQ水
    注意:这里使用的烧杯或瓶子不是用肥皂洗涤的,因为大多数商业肥皂的钡含量。
  17. 表型抗体(用0.1μm旋转过滤器过滤)(Millipore,目录号:UFC30VV00)
  18. Ir-嵌入剂储备溶液(Fluidigm,目录号:201192)
    注意:可以使用Rh103-嵌入剂。
  19. 10×基于皂苷的透化缓冲液(eBioscience,目录号:00 8333-56)
  20. 佛波醇12-豆蔻酸酯13-乙酸酯(PMA)(Sigma-Aldrich,目录号:P8139)
  21. Ionomycine(Sigma-Aldrich,目录号:I0634)
  22. 植物凝集素(PHA)(Sigma-Aldrich,目录号:61764)
  23. SEB(Sigma-Aldrich,目录号:S0812)
  24. 抗CD3/CD28(各种供应商)
  25. 肽混合物(JPT)
  26. 完成RPMI(参见配方)
  27. CyPBS(参见配方)
  28. CyFACS缓冲区(参见配方)
  29. 活死皮(见配方)

设备

  1. 96孔圆底板
  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

程序

  1. 解冻PBMC
    1. 将温热的完全RPMI培养基在37℃水浴中。 每个样品将 需要22ml含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(终浓度25U/ml)。 加入Benzonase 进入培养基以防止死细胞聚集。 解冻不超过3 样品。 对每批样品运行一个控制PBMC
    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. 以1550rpm(RCF = 473)在室温下离心细胞8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。 加9ml以上温热 benzonase培养基加到管中
    11. 以1550rpm离心细胞(RCF =   473)在室温下孵育8分钟。 从细胞中去除上清液 并通过点击管重新悬浮颗粒
    12. 将细胞重悬于1ml温热培养基中
    13. 计数细胞与Vicell(或如果必要的血细胞计数器)。 计数, 取20微升细胞,并用480微升PBS在Vicell计数室中稀释。 作为PBMC以1:25稀释倍数加载到Vicell上
    14. 用温热培养基(此时不再有benzonase)将细胞浓度调节至5-10×10 6个细胞/ml。
    15. 使用多通道移液器,添加200微升细胞(至少1 x 10 6细胞)加入96孔深孔板的每个孔中。 拆分每个 样品平均地进入两个或更多个孔,保持一个未刺激 控制和其他不同类型的刺激
    16. 在37℃下在CO 2培养箱中休息过夜(6-18小时)

  2. 刺激细胞
    1. 在37℃下过夜休息后,加入活化试剂和分泌物   抑制剂(布雷菲德菌素A /莫能菌素)加入到孔中用于刺激。 仅添加 分泌抑制剂到未刺激的对照孔。 如果做 CD107a染色,在刺激期间加入CD107a抗体
      表1.蛋白质分泌抑制剂
      试剂
      库存量
      中间稀释
      最终浓度
      布雷菲德菌素A
      5mg/ml的DMSO溶液 (以等分试样在-20℃下储存) 1:10在PBS
      10μg/ml(1:50)或5μg/ml(1:100)
      莫能菌
      5mg/ml的乙醇溶液 (储存于-20℃)
      1:10在PBS
      10μg/ml(1:50)或5μg/ml(1:100)的布雷菲德菌素

      表2.激活器
      试剂
      库存量
      中间稀释
      最终浓度
      佛波醇12-豆蔻酸酯13-乙酸酯(PMA)
      1mg/ml的DMSO溶液 (以等分试样在-20℃下储存) 1:1,000在PBS中
      10 ng/ml
      益生霉素
      1mg/ml的DMSO溶液 (以等分试样在-20℃下储存) 1:10在PBS中
      1μg/ml
      植物凝集素(PHA)
      1mg/ml的DMSO溶液 (储存在4℃)
      1:10在PBS
      1μg/ml
      SEB
      50μg/ml,在PBS中

      1μg/ml(1:50)

      抗CD3/CD28
      按照制造商说明
      -
      -
      肽混合物
      0.5-1mg/ml/pep的DMSO溶液 (以等分试样在-20℃下储存) 1:10在PBS
      1μg/ml /肽
      (1:50 - 1:100)

      注意:
      1. 避免溶剂毒性是重要的。 最终DMSO +乙醇 浓度从所有来源(肽,布雷菲德菌素A,莫能菌素)应该 不超过0.5%。
      2. 对大多数细胞因子:使用10μg/ml的布雷菲德菌素A. 最终浓度(见储备制备表)。 对于CD107和CD154: 使用最终浓度为10μg/ml的莫能菌素(见制备物 表)。 对于组合细胞因子和CD107或CD154的测定:使用布雷菲德菌素   A和莫能菌素,每种终浓度为5μg/ml。
      3. 金属标记的CD107和CD154可以在培养过程中加入 以2μg/ml的浓度刺激。 这允许染色 在活化期间被细胞再内化的靶分子   过程。
      4. 添加共刺激抗体是任选的。 添加1   μg/ml的最终浓度的CD28和/或CD49d(标记的抗体可以是   如果需要分析标记,则使用)。
    2. 孵育细胞 (PMA +离子霉素刺激,PHA +离子霉素刺激)或4小时 6-8小时(SEB,抗CD3/CD28刺激,肽刺激)在37℃,   CO 2培养箱。
      注意:对于大多数细胞因子,在37℃下孵育6-12小时是足够的; 对于IL-10最佳孵化时间为12-24小时,但它   可能在6小时内检测到。
    3. 在刺激结束时,加入EDTA至终浓度为2mM,并在室温下孵育15分钟。

  3. 染色
    1. 每孔用250μlCyFACS缓冲液洗涤2次,每孔1550rpm(RCF = 473),10分钟   。 使用相同的体积和离心条件   在用PFA固定之前的额外的洗涤步骤(步骤C6)
    2. 在CyFACS缓冲液中制备表面Ab混合物(用0.1μm旋转过滤器过滤 过滤器)每反应100μl。 在冰上孵育45分钟。 使用供应商 推荐浓度(或为自制的最佳滴度 缀合物)
    3. 在CyFACS缓冲液中洗涤2x
    4. 重悬细胞在100μl的1:3,000稀释5毫克/毫升115-DOTA马来酰亚胺 (滴定,如果新股票)在CyPBS,在冰上孵育30分钟
    5. 在CyFACS缓冲液中洗涤3次
    6. 重悬于100μl2%PFA的CyPBS,孵育4℃过夜
    7. 在1x eBioscience perm缓冲液(1x,milliQ水)中洗涤2x,2,000 rpm(RCF = 787),4℃10分钟。 相同体积和离心机 条件用于以下洗涤步骤。
    8. 使 细胞内染色混合物在1x perm缓冲液中并用0.1μm过滤 旋转过滤器,每个反应100μl。 在冰上孵育45分钟。
    9. 在CyFACS缓冲液中洗涤3次
    10. 重悬于稀释在2%PFA(在CyPBS中)的100μl1:2000的Ir-螯合剂中。
    11. 在室温孵育20分钟。
    12. 在CyFACS缓冲液中洗涤2x
    13. 在MilliQ水中洗3次。
    14. 重悬于MilliQ水(1至1.5 ml)中,用于在CyTOF上运行

代表数据



图1. PMA/Ionomycin刺激后的细胞内标记物的流动门控

食谱

  1. 完成RPMI
    RPMI1640
    10%FBS
    1x pen-strep-Glutamine
  2. CyPBS
    1x PBS,不含重金属污染物,例如10x PBS
    在MilliQ水中制造
  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%指定同位素;痕量金属纯99.99%)

致谢

这项工作得到国家卫生研究院的拨款#S10RR027582和5U19AI057229的支持。我们感谢Evan Newell和Mark Davis在初始协议开发方面的帮助。

参考文献

  1. 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。
  2. 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。 科学 332(6030):687-
  3. Maecker,H.T。(2009)。 多参数流式细胞术监测T细胞反应。 em> 485:375-391。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lin, D., Gupta, S. and Maecker, H. (2015). Intracellular Cytokine Staining on PBMCs Using CyTOFTM Mass Cytometry. Bio-protocol 5(1): e1370. DOI: 10.21769/BioProtoc.1370.
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婷 王
天津医科大学总医院
9/4/2017 2:25:59 PM Reply
Holden Maecker
Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, USA

Thanks for your interest in the protocol. You can use either CD3/CD28 or PMA/ionomycin to stimulate Th1, Th2, and Th17 cytokine secretion from T cells. The difference is that PMA/ionomycin will stimulate many more T cells, as well as NK cells, monocytes, etc. With CD3/CD28, a smaller fraction of T cells respond, and you may not see the more rare cytokines like IL-4.

9/4/2017 10:27:04 AM