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Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs)
人淋巴细胞或外周血单核细胞(PBMC)的细胞因子胞内染色(ICS)   

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Abstract

Production of cytokines plays an important role in the immune response. Cytokines are involved in many different pathways including the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha. Cytokines are not preformed factors but are rapidly produced and secreted in response to cellular activation. Intracellular cytokine detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing direct TH1 versus TH2 determination. It can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or with MHC multimers to detect an antigen specific response, making it an extremely flexible and versatile method. This capability, combined with the high throughput nature of the instrumentation, gives intracellular cytokine staining an enormous advantage over existing single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The principle steps of intracellular cytokine staining is as follows:
1. Cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail;
2. An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the cytokines within the cell;
3. Next, EDTA is added to remove adherent cells from the activation vessel;
4. After washing, antibodies to cell surface markers can be added to the cells;
5. The cells are then fixed in paraformaldehyde and permeabilized;
6. The anti-cytokine antibody is added and the cells can be analyzed by flow cytometer.

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. 100x Pen-strep-Glutamine (Hyclone, catalog number: SV30082.01 )
  5. Benzonase (Sigma-Aldrich, catalog number: B7651 )
  6. PBS (10x stock) (Rockland, catalog number: MB-008 )
  7. Sodium azide (10% w/v solution) (Teknova, catalog number: S0209 )
  8. PMA (Sigma-Aldrich, catalog number: P8139 )
  9. Ionomycin (Calbiochem®, catalog number: 407952 )
  10. Dynabeads Human T Activator CD3/CD28 (Life Technologies, InvitrogenTM, catalog number: 111.32D )
  11. Brefeldin A (Sigma-Aldrich, catalog number: B7651)
  12. 1,000x monensin (BioLegend, catalog number: 420701 )
  13. 0.5 M EDTA (Sigma-Aldrich, catalog number: E-5134 )
  14. LIVE/DEAD® fixable red dead cell stain (Life Technologies, InvitrogenTM, catalog number: L23102 )
  15. 10x FACS lysing solution (BD Biosciences, catalog number: 349202 )
  16. 10x FACS permeabilizing solution 2 (BD Biosciences, catalog number: 347692 )
  17. Fluorochrome-linked surface markers [e.g. CD3-V500 (BD Biosciences, catalog number: 561416 ); CD8-V450 (BD Biosciences, catalog number: 560348 ); CD4-PerCP-Cy5.5 (BD Biosciences, catalog number: 341654 )]
  18. Fluorochrome-linked cytokine antibodies [e.g. IFN gamma-FITC (BD Biosciences, catalog number: 340449 ); IL-17 PE (BD Biosciences, catalog number: 560438 ); IL-2 PE-Cy7 (BD Biosciences, catalog number: 560707 ); IL-22-APC (R&D systems, catalog number: IC7821A); TNF- Alexa fluor 700 (BD Biosciences, catalog number: 557996 )]
  19. BD CompBeads [(anti-mouse Igκ, anti-rat Igκ, or anti-rat/hamster Igκ; BD Biosciences), for creating single-color compensation controls (BD Biosciences, catalog number: 560707)]
  20. Immunoglobulin capture beads for single-color compensation (e.g., BD Biosciences, catalog number: 560497 )
  21. Complete RPMI (see Recipes)
  22. FACS buffer (see Recipes)

Equipment

  1. 96- deep well V-bottom plates (Corning, catalog number: 3960 ) (1 ml washes in a 2 ml well volume)
  2. Falcon round-bottom FACS tubes
  3. Magnet for Dynabead separation (BD Biosciences, IMag, catalog number: 552311 )
  4. 37 °C water bath
  5. Biosafety cabinet
  6. Centrifuge
  7. CO2 incubator at 37 °C
  8. Calibrated pipettes
  9. ViCell (Beckman Coulter) or Hemocytometer cell counter

Procedure

  1. Thaw PBMC
    1. Warm complete medium to 37 °C in water bath. Each sample will require 22 ml of medium with benzonase. Calculate the amount needed to thaw all samples, and prepare a separate aliquot of warm medium with 1:10,000 benzonase (final concentration: 25 U/ml). Thaw no more than 3 samples at a time.
      Note: Run one control PBMC with each batch of samples if doing a large longitudnal study split over sevral batches.
    2. Remove samples from liquid nitrogen and transport to lab on dry ice.
    3. Place 10 ml of warmed benzonase medium 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 medium from appropriately labeled centrifuge tube slowly to the cells, then transfer the cells to the centrifuge tube. Rinse vial with more medium 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), 10 min at room temperature.
    9. Decant or aspirate supernatant from the cells and resuspend the pellet by tapping the tube.
    10. Gently resuspend the pellet in 1 ml warmed benzonase medium. Filter cells through a 70 μm cell strainer if needed. Add 9 ml more warmed benzonase medium to the tube.
    11. Centrifuge cells at 1,550 rpm (RCF = 473), 10 min at room temperature. Decant or aspirate supernatant from the cells and resuspend the pellet by tapping the tube.
    12. Resuspend cells in 1 ml warm medium.
    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 medium (no more benzonase at this point).
    15. Using a multichannel pipette, add 200 µl cells (1 x 106 cells) into each well of a 96-well deep well plate. Split each sample 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.
      Note: The percentage of both CD4+ and CD8+ cytokine producing T cells is increased after an overnight resting prior to stimulation (Horton et al., 2007).

  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.

      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 4 °C)
      1:10 in PBS
      10 μg/ml (1:50) or
      5 μg/ml (1:100) with brefeldin A

      Table 2. Activators
      Reagent
      Stock concentration
      Intermediate dilution
      Final concentration
      Phorbol12-myristate13 acetate (PMA)
      1 mg/ml in DMSO
      (stored in aliquots at -20 °C)
      1:1,000 in PBS
      10 ng/ml
      Ionomycin
      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
      Peptide mixes
      0.5-1 mg/ml/pep in DMSO (stored in aliquots at -20 °C)
      1:10 in PBS
      1 μg/ml/peptide
      Anti-CD3/CD28
      Follow manufacturer instructions
      -
      -

      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. Fluorochrome-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. These can increase the cytokine response to protein antigens, peptides, and SEB by amplifying the signal for low-affinity T cells (Waldrop et al., 1998). 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 and TGF optimal incubation time is 12-24 h.
    3. Add EDTA to a final concentration of 2 mM and incubate for 15 min at room temperature.
      Note: If Dynabeads have been used for stimulation, use the I Mag for removal of beads during this step.
    4. Wash the cells with PBS at 1,550 rpm (RCF = 473), 8 min at room temperature.

  3. Staining
    1. Repeat PBS wash at 1,550 rpm (RCF = 473), 8 min, room temperature and resuspend the cells in 500 μl PBS.
    2. Stain with Red LIVE/DEAD cell viability dye according to the manufacturer's instructions.
    3. Incubate at room temperature for 30 min in dark.
    4. Centrifuge the cells at 1,550 rpm (RCF = 473), 10 min, room temperature.
    5. Flick or aspirate to remove supernatant and wash cells with PBS at 1,550 rpm (RCF = 473), 10 min, room temperature.
    6. Repeat another wash in FACS buffer, at 1,550 rpm (RCF = 473), 10 min, room temperature.
    7. Flick or aspirate to remove supernatant and resuspend cells in residual volume.
    8. Prepare the surface staining cocktail according to titre per test for each antibody (pre-determined/provided by manufacture). See an example below for staining ten tubes/wells.
      Note: The manufacturer’s recommended dose is a good starting point, but antibody titrations on representative cells can improve results on unsatisfactory stainings.

      Table 3. Surface staining cocktail
      Antibody panel
      Stain
      Titre (μl/sample)
      X # of samples
      Total μl
      CD3
      V 500
      5
      10
      50
      CD4
      PerCPCy 5.5
      10
      10
      200
      CD8
      V 450
      5
      10
      50
      Note: If doing simultaneous tetramer analysis, cells should be stained with the pMHC multimer prior to staining with the anti-coreceptor antibodies, in a separate step, washed and then stained with the surface staining cocktail.

    9. Add calculated volume of staining cocktail for each sample to (20 μl in the example above). Add appropriate amount of single antibodies to beads for compensation controls.
    10. Incubate cells for 30 min at room temperature in the dark.
    11. Add 2 ml FACS buffer to each well/tube.
    12. Centrifuge the cells at 1,550 rpm (RCF = 473), 10 min, room temperature.
    13. Flick or aspirate to remove supernatant and wash cells again with FACS buffer at 1,550 rpm (RCF = 473), 10 min, room temperature.
    14.  Prepare 1x BD FACS lysing solution (10x diluted to 1x in water).
    15.  Add 2 ml per tube/well, mix well and incubate the cells for 10 min at room temperature, in the dark.
    16. Centrifuge cells at 2,000 rpm (RCF = 787) for 10 min at 4 °C and flick or aspirate to remove supernatant.
    17. Wash cells twice in FACS buffer.
    18. Prepare 1x BD FACS Perm-2 buffer (10x diluted to 1x in water).
    19. Resuspend the cell pellet in 500 μl of 1x FACS permeabilizing solution (prepared above) and incubate for 15 min in dark.
    20. Centrifuge cells at 2,000 rpm (RCF = 787) for 10 min and flick or aspirate to remove supernatant.
    21. Wash cells twice in FACS buffer.
    22. Flick or aspirate to remove supernatant and resuspend cells in residual volume.
    23. Prepare the intracellular staining cocktail according to titre per test for each antibody (pre-determined/provided by manufacture). See example below for staining ten tubes/wells.

      Table 4. Intracellular staining cocktail
      Antibody panel
      Stain
      Titre(μl/ sample)
      X # of samples
      Total μl
      IFN-γ
      FITC
      20
      10
      200
      IL-17
      PE
      20
      10
      200
      IL-4
      APC
      5
      10
      50
      TNFα
      Alexa700
      1.2
      10
      12
      IL-2
      PE-Cy7
      5
      10
      50

    24. Calculate and add required volume of staining cocktail to each sample (51.2 μl in the example above) and incubate cells for 30 min, in the dark.
    25. Add 2 ml FACS buffer to each well/tube.
    26. Centrifuge cells at 2,000 rpm for 10 min and flick or aspirate to remove supernatant.
    27. Wash cells twice in FACS buffer.
    28. Flick or aspirate to remove supernatant and resuspend in a final volume of 150 μl FACS buffer for Flow.

Representative data



Figure 1. Intracellular cytokine production by T cells (IFNg, TNF, IL-2, IL-17, IL-22). The first three plots in each row show gating hierarchy for CD4+ and CD8+ T cells. The next panel of subsequent three plots shows cytokine production by CD4+ T cells and the final panel shows the same for CD8+ T cells. (A) unstimulated (B) PMA and Ionomycin stimulation (C) PHA and Ionomycin stimulation (D) anti CD3/CD28 stimulation.

Notes

  1. Doing the adequate number of washes after staining, fixing and permeabilization of cells is very important for reducing the background level for cytokines.
  2. If doing the experiment in a 96-well plate, leave a few wells empty between the unstimulated and stimulated conditions to minimize the chances of spillover and cross- contamination while washing.

Recipes

  1. Complete RPMI
    RPMI
    10% serum (e.g. FBS)
    Pen-strep
    Glutamine
  2. FACS buffer
    PBS with 2% serum (e.g. FBS) and 0.1% Na azide

References

  1. Horton, H., Thomas, E. P., Stucky, J. A., Frank, I., Moodie, Z., Huang, Y., Chiu, Y. L., McElrath, M. J. and De Rosa, S. C. (2007). Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods 323(1): 39-54.
  2. Jung, T., Schauer, U., Heusser, C., Neumann, C. and Rieger, C. (1993). Detection of intracellular cytokines by flow cytometry. J Immunol Methods 159(1-2): 197-207.
  3. Lovelace, P., and Maecker, H. T. (2010). In: Hawley, T. S. and Hawley, R. G. (eds). Multiparameter intracellular cytokine staining. Humana Press, 165-178.
  4. Nylander, S. and Kalies, I. (1999). Brefeldin A, but not monensin, completely blocks CD69 expression on mouse lymphocytes: efficacy of inhibitors of protein secretion in protocols for intracellular cytokine staining by flow cytometry. J Immunol Methods 224(1-2): 69-76.
  5. Pitcher, C. J., Quittner, C., Peterson, D. M., Connors, M., Koup, R. A., Maino, V. C. and Picker, L. J. (1999). HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression. Nat Med 5(5): 518-525.
  6. Waldrop, S. L., Davis, K. A., Maino, V. C. and Picker, L. J. (1998). Normal human CD4+ memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis. J Immunol 161(10): 5284-5295.

简介

细胞因子的产生在免疫应答中起重要作用。细胞因子参与许多不同的途径,包括通过IFNγ诱导许多抗病毒蛋白,通过IL-2诱导T细胞增殖以及抑制病毒基因表达和TNFα的复制。细胞因子不是预先形成的因子,但是响应于细胞激活而快速产生和分泌。通过流式细胞术的细胞内细胞因子检测已经成为研究单细胞水平的细胞因子产生的首要技术。它检测细胞刺激后细胞因子在内质网内的产生和积累,允许直接TH1对TH2测定。它也可以与其他流式细胞术协议结合使用细胞表面标记或与MHC多聚体进行免疫分型,以检测抗原特异性反应,使其成为一种非常灵活和通用的方法。这种能力,结合仪器的高通量性质,使细胞内细胞因子染色与现有的单细胞技术如ELISPOT,有限稀释和T细胞克隆相比具有巨大的优势。细胞内细胞因子染色的主要步骤如下:
1。使用特异性肽或非特异性活化混合物将细胞活化几小时;
2。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;
4。洗涤后,可将细胞表面标记的抗体加入细胞中;
5。然后将细胞固定在多聚甲醛中并透化;
6。加入抗细胞因子抗体,并且可以通过流式细胞仪分析细胞。

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. 100x Pen-strep-Glutamine(Hyclone,目录号:SV30082.01)
  5. Benzonase(Sigma-Aldrich,目录号:B7651)
  6. PBS(10x储备液)(Rockland,目录号:MB-008)
  7. 叠氮化钠(10%w/v溶液)(Teknova,目录号:S0209)
  8. PMA(Sigma-Aldrich,目录号:P8139)
  9. 离子霉素(Calbiochem ,目录号:407952)
  10. Dynabeads人T激活剂CD3/CD28(Life Technologies,Invitrogen TM ,目录号:111.32D)
  11. 布雷菲德菌素A(Sigma-Aldrich,目录号:B7651)
  12. 1,000x莫能菌素(BioLegend,目录号:420701)
  13. 0.5M EDTA(Sigma-Aldrich,目录号:E-5134)
  14. LIVE/DEAD 可固定红色死细胞染色剂(Life Technologies,Invitrogen TM ,目录号:L23102)
  15. 10x FACS裂解液(BD Biosciences,目录号:349202)
  16. 10x FACS透化溶液2(BD Biosciences,目录号:347692)
  17. 荧光染料连接的表面标记[例如CD3-V500(BD Biosciences,目录号:561416); CD8-V450(BD Biosciences,目录号:560348); CD4-PerCP-Cy5.5(BD Biosciences,目录号:341654)]
  18. 荧光染料连接的细胞因子抗体[例如IFNγ-FITC(BD Biosciences,目录号:340449); IL-17PE(BD Biosciences,目录号:560438); IL-2 PE-Cy7(BD Biosciences,目录号:560707); IL- 22-APC(R& D systems,目录号:IC7821A); TNF-Alexa fluor 700(BD Biosciences,目录号:557996)]
  19. BD CompBeads [(抗小鼠Igκ,抗大鼠Igκ或抗大鼠/仓鼠Igκ; BD Biosciences)用于产生单色补偿对照(BD Biosciences,目录号:560707)]
  20. 用于单色补偿的免疫球蛋白捕获珠子(例如,BD Biosciences,目录号:560497)
  21. 完成RPMI(参见配方)
  22. FACS缓冲区(参见配方)

设备

  1. 96-深孔V底板(Corning,目录号:3960)(在2ml孔体积中洗涤1ml)
  2. Falcon圆底FACS管
  3. 用于Dynabead分离的磁铁(BD Biosciences,IMag,目录号:552311)
  4. 37°C水浴
  5. 生物安全柜
  6. 离心机
  7. CO 2培养箱中37℃培养
  8. 校准移液器
  9. ViCell(Beckman Coulter)或血细胞计数器计数器

程序

  1. 解冻PBMC
    1. 温热完全培养基至37℃水浴。 每个样品将需要22   ml具有benzonase的培养基。 计算解冻所需的金额 样品,并用1:10,000制备单独的温热培养基等分试样 benzonase(终浓度:25U/ml)。 解冻不超过3个样品   一个时间。
      注意:如果进行大量纵向研究分成两批,则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. 加入1毫升温暖的benzonase培养基从适当标记 离心管慢慢地将细胞转移到细胞中 离心管。 用更多的介质从离心管冲洗小瓶 检索所有单元格。
    7. 尽快继续其余样品。
    8. 以1550rpm(RCF = 473)离心细胞,在室温下离心10分钟
    9. 倾析或吸出细胞的上清液,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培养基到管中
    11. 以1550rpm离心细胞 (RCF = 473),在室温下10分钟。 倾析或吸出上清液 从细胞和通过轻拍管重悬颗粒
    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,加入200微升细胞(1×10 6个细胞)   每孔96孔深孔板。 将每个样品分成两个或 更多的井保持一个作为未刺激的控制和其他 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) 注意:CD4 + 和CD8 /sup> 细胞因子产生性T细胞 在刺激后过夜休息后增加(Horton et al。,2007)。

  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。

      表1.蛋白质分泌抑制剂
      试剂
      库存集中
      中级稀释
      最终集中
      布雷菲德菌素A
      在DMSO中5mg/ml(以等分试样在-20℃下储存) 1:10在PBS
      10μg/ml(1:50)或
      5μg/ml(1:100)和莫能菌素
      莫能菌
      5mg/ml的乙醇溶液(4℃保存) 1:10在PBS
      10μg/ml(1:50)或
      5μg/ml(1:100)和布雷菲德菌素A/B

      表2.激活器
      试剂
      库存集中
      中级稀释
      最终集中
      佛波醇12-肉豆蔻酸酯13(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
      离子霉素
      1mg/ml的DMSO溶液 (以等分试样在-20℃下储存) 1:10在PBS
      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
      肽混合物
      0.5-1mg/ml/pep于DMSO中(以等分试样储存于-20℃) 1:10在PBS
      1μg/ml /肽
      抗CD3/CD28
      按照制造商说明
      -
      -

      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. 可以将荧光染料标记的CD107和CD154加入培养物中 在浓度为2μg/ml的刺激期间。 这允许 染色在细胞期间再内化的靶分子 激活过程。
      4. 添加共刺激抗体是 可选的。 这些可以增加细胞因子对蛋白质抗原的反应, 肽和SEB,通过扩增低亲和力T细胞的信号 (Waldrop等人,1998)。 加入1μg/ml终浓度的CD28和/或 如果标记的分析是,可以使用CD49d(标记的抗体 需要)。
    2. 孵育细胞4小时(PMA +离子霉素 刺激,PHA +离子霉素刺激)或6-8小时(SEB,抗CD3/CD28 刺激,肽刺激)在37℃下在CO 2培养箱中培养。
      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和TGF的最佳培养时间为12-24小时。
    3. 加入EDTA至终浓度为2mM,并在室温下孵育15分钟 注意:如果Dynabeads已被用于刺激,请在此步骤中使用I Mag去除珠粒。
    4. 用PBS以1,550rpm(RCF = 473)洗涤细胞,在室温下洗8分钟。

  3. 染色
    1. 重复PBS洗涤在1,550 rpm(RCF = 473),8分钟,室温和重悬细胞在500μlPBS。
    2. 根据制造商的说明用红色LIVE/DEAD细胞活力染料染色。
    3. 在室温下避光孵育30分钟。
    4. 以1550rpm(RCF = 473℃)离心细胞,10分钟,室温
    5. 轻轻或吸出以除去上清液,并用PBS以1,550rpm(RCF = 473),10分钟,室温洗涤细胞。
    6. 在FACS缓冲液中以1550rpm(RCF = 473)重复另一次洗涤,10分钟,室温。
    7. 轻轻或吸出以除去上清液,并在剩余体积中重悬细胞
    8. 根据每个测试的滴度制备表面染色混合物 (预定/由制造商提供)。 参见 下面的例子是染色十个管/孔 注意:制造商的   推荐剂量是一个很好的起点,但抗体滴定开 代表性细胞可以提高对不良染色的结果。

      表3.表面染色鸡尾酒
      抗体面板
      污渍
      滴定(μl/样品)
      X个样品
      总μl
      CD3
      V 500
      5
      10
      50
      CD4
      perCPCy 5.5
      10
      10
      200
      CD8
      V 450
      5
      10
      50
      注意:如果进行同时四聚体分析,在单独的步骤中用抗 - 共同受体抗体染色之前,细胞应用pMHC多聚体染色,洗涤,然后用表面染色混合物染色。
      />
    9. 将每个样品的计算体积的染色混合物加入(20μl   在上面的例子中)。 加入适量的单一抗体 珠补偿控制。
    10. 在室温下在黑暗中孵育细胞30分钟
    11. 向每个孔/管中加入2ml FACS缓冲液
    12. 以1550rpm(RCF = 473℃)离心细胞,10分钟,室温
    13. 轻拂或吸出以除去上清液并再次用细胞洗涤细胞 FACS缓冲液以1550rpm(RCF = 473℃),10分钟,室温
    14.  准备1x BD FACS裂解溶液(在水中10x稀释至1x)。
    15.  每管加入2毫升/孔,混匀,并在室温下,在黑暗中孵育细胞10分钟。
    16. 在4℃下以2,000rpm(RCF = 787)离心细胞10分钟,轻弹或吸出以除去上清液。
    17. 在FACS缓冲液中洗涤细胞两次
    18. 准备1x BD FACS Perm-2缓冲液(10x在水中稀释至1x)
    19. 将细胞沉淀重悬在500μl1×FACS透化溶液(上面制备)中,并在暗处孵育15分钟。
    20. 离心细胞在2000 rpm(RCF = 787)10分钟,轻弹或吸出去除上清。
    21. 在FACS缓冲液中洗涤细胞两次
    22. 轻轻或吸出以除去上清液,并在剩余体积中重悬细胞
    23. 根据滴度制备细胞内染色混合物   测试每种抗体(预定/由制造商提供)。 看到 下面的例子用于染色10个管/孔。

      表4.细胞内染色混合物
      抗体面板
      污渍
      滴定(μl/样品)
      X个样品
      总μl
      IFN-γ
      FITC
      20
      10
      200
      IL-17
      PE
      20
      10
      200
      IL-4
      APC
      5
      10
      50
      TNFα
      Alexa700
      1.2
      10
      12
      IL-2
      PE-Cy7
      5
      10
      50

    24. 计算并向每个添加所需体积的染色混合物 样品(在上述实施例中为51.2μl)并将细胞孵育30分钟, 黑暗。
    25. 向每个孔/管中加入2ml FACS缓冲液
    26. 离心细胞在2,000 rpm离心10分钟,轻弹或吸出去除上清
    27. 在FACS缓冲液中洗涤细胞两次
    28. 轻轻或吸出以除去上清液,并在最终体积为150μl用于Flow的FACS缓冲液中重悬。

代表数据



图1.T细胞(IFNg,TNF,IL-2,IL-17,IL-22)的细胞内细胞因子产生。每行中的前三个图显示CD4 和CD8 + T细胞。接下来的三个图的下一组显示了CD4 + T细胞的细胞因子产生,最后一组显示了CD8 + T细胞的细胞因子产生。 (A)未刺激(B)PMA和离子霉素刺激(C)PHA和离子霉素刺激(D)抗CD3/CD28刺激。

笔记

  1. 在染色,固定和透化细胞后进行足够数量的洗涤对于降低细胞因子的背景水平是非常重要的。
  2. 如果在96孔板中进行实验,在未刺激和刺激条件之间留出几个孔,以尽量减少洗涤时溢出和交叉污染的机会。

食谱

  1. 完成RPMI
    RPMI
    10%血清(例如FBS)
    Pen-strep
    谷氨酰胺
  2. FACS缓冲区
    具有2%血清(例如FBS)和0.1%叠氮化钠的PBS

参考文献

  1. Horton,H.,Thomas,E.P.,Stucky,J.A.,Frank,I.,Moodie,Z.,Huang,Y.,Chiu,Y.L.,McElrath,M.J.and De Rosa, 优化和验证8色细胞内细胞因子染色(ICS)测定以定量抗原特异性T由免疫接种诱导的细胞。 Immunol Methods 323(1):39-54。
  2. Jung,T.,Schauer,U.,Heusser,C.,Neumann,C.and Rieger,C。(1993)。 通过流式细胞术检测细胞内细胞因子。 J Immunol方法 159(1-2):197-207。
  3. Lovelace,P.,和Maecker,H.T。(2010)。参见:Hawley,T.S。和Hawley,R.G。(eds)。多参数细胞内细胞因子染色。 Humana Press,165-178
  4. Nylander,S。和Kalies,I。(1999)。 Brefeldin A,但不是莫能菌素,完全阻断CD69在小鼠淋巴细胞上的表达:蛋白质分泌抑制剂的功效在通过流式细胞术进行细胞内细胞因子染色的方案中。< j> J Immunol Methods 224(1-2):69-76。
  5. Pitcher,C.J.,Quittner,C.,Peterson,D.M.,Connors,M.,Koup,R.A.,Maino,V.C.and Picker,L.J。(1999)。 HIV-1特异性CD4 + T细胞在具有活性HIV-1感染的大多数个体中是可检测的,但随着病毒抑制的延长而下降。 Nat Med 5(5):518-525。
  6. Waldrop,S.L.,Davis,K.A.,Maino,V.C。和Picker,L.J。(1998)。 正常人CD4 + 记忆T细胞在其活化阈值中显示出广泛的异质性 用于细胞因子合成。 Immunol 161(10):5284-5295。
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引用:Gupta, S. and Maecker, H. (2015). Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs). Bio-protocol 5(7): e1442. DOI: 10.21769/BioProtoc.1442.
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xiaoyun lin
Tianjin Medical University
1.what degree can polarization inducing influence the intracellular cytokine expression before PMA/ionomycin stimulating. 2.Is there any relation between the activator concentration and different cytokine expression. 3.Could this protocol apply to a mice PBMCs smaple.
10/29/2017 6:36:44 AM Reply
Holden Maecker
Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, USA

Short-term stimulation with PMA+ionomycin should not affect polarization of cells to Th1, Th2, etc. it should just reveal the existing potential of the cells. Similarly, I don't think stimulus concentration should differentially affect cytokine readouts, though we haven't rigorously tested this. The protocol should be adaptable to mouse cells, with appropriate mouse-specific antibodies.

10/29/2017 8:54:26 AM