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Intracellular Cytokine (INF-gamma) Staining Assay
细胞因子(INF-gamma)的胞内染色方法   

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Abstract

An intracellular cytokine (INF-gamma) staining assay is used to analyze the function of lymphocytes at the single cell level. By combining surface staining and intracellular cytokine staining, this assay can reveal the percentage of cytokine-releasing cells in a particular population, which cannot be obtained from an ELISpot assay.

Materials and Reagents

  1. PE Rat Anti-Mouse IFN-γ (BD Biosciences, catalog number: 554412 )
  2. PE Rat IgG1 κ Isotype Control (BD Biosciences, catalog number: 554685 )
    Note: The above antibodies have been tested by the author and may be substituted with the antibodies desired by users.
  3. BD Cytofix/CytopermTM Fixation/Permeabilization Solution Kit with BD GolgiStopTM (BD Biosciences, catalog number: 554715 )
  4. 1x phosphate buffered saline (PBS)
  5. FACS staining buffer (1x PBS, 2% FBS)
  6. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
  7. Ionomycin (Sigma-Aldrich, catalog number: I9657 )

Equipment

  1. BECKMAN centrifuges and rotor (Beckman Coulter)
  2. Incubator
  3. 96 well plates
  4. Falcon round-bottom tubes

Procedure

  1. Stimulation of cells
    1. For stimulating IFN-gamma release from T cells, add PMA (5 ng/ml) and ionomycin (500 ng/ml) to the cell culture and incubate for 6 h at 37 °C.
    2. Add 4 µl of BD GolgiStopTM for every 6 ml of cell culture and mix thoroughly (it is recommended that BD GolgiStop not be kept in cell culture for longer than 12 h).

  2. Harvest cells
    1. Collect the cells and centrifuge at 1,500 rpm for 3 min.
    2. Wash twice with PBS.
    3. Dilute single-cell suspensions to 1 x 107 cells/ml in PBS with 2% FBS.
    4. Add 100 µl cells (1 x 106) per well in 96 well plates.
    5. Spin plate at 800 x g, 3 min, at 4 °C.
    6. Wash 3 times with cold PBS, spinning as in step 7.

  3. Stain cell surface antigens
    1. Resuspend the cells in 100 µl Fc block (recommended dilution: 1:1,000 in PBS/2% FBS). Incubate on ice, 10 min. Spin.
    2. Resuspend in 100 µl surface antibody mixture (recommend dilution: 1:100 in PBS/2% FBS). Incubate at room temperature, 20 min in the dark. Spin.
    3. Wash once with cold PBS.

  4. Stain intracellular antigens
    1. Resuspend in 200 µl of BD Cytofix/CytoPerm solution. Incubate at room temperature, 30 min in the dark. Spin 1500 x g, 3 min, 4 °C.
    2. Wash twice with 200 µl BD Perm/wash buffer. Spin as in step 12.
    3. Resuspend in 100 µl cytokine stain (recommended dilution: 1:100 in 1x Perm/Wash). Incubate on ice, 30 min in the dark. Spin as in step 12.
    4. Wash twice with BD Perm/Wash, spinning as in step 12.
    5. Resuspend cells in 300-400 µl FACS buffer and transfer to Falcon round-bottom tubes for acquisition on a flow cytometer.

Acknowledgments

This protocol was previously used in Assenmacher et al. (1994) and Shang et al. (2004).

References

  1. Assenmacher, M., Schmitz, J. and Radbruch, A. (1994). Flow cytometric determination of cytokines in activated murine T helper lymphocytes: expression of interleukin-10 in interferon-gamma and in interleukin-4-expressing cells. Eur J Immunol 24(5): 1097-1101.
  2. Shang, X. Y., Chen, H. S., Zhang, H. G., Pang, X. W., Qiao, H., Peng, J. R., Qin, L. L., Fei, R., Mei, M. H., Leng, X. S., Gnjatic, S., Ritter, G., Simpson, A. J., Old, L. J. and Chen, W. F. (2004). The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients. Clin Cancer Res 10(20): 6946-6955.

简介

细胞内细胞因子(INF-γ)染色测定用于分析淋巴细胞在单细胞水平的功能。 通过组合表面染色和细胞内细胞因子染色,该测定可以揭示特定群体中细胞因子释放细胞的百分比,其不能从ELISpot测定获得。

材料和试剂

  1. PE大鼠抗小鼠IFN-γ(BD Biosciences,目录号:554412)
  2. PE大鼠IgG1κ同种型对照(BD Biosciences,目录号:554685) 注意:上述抗体已经被作者测试,可能被用户需要的抗体替代。
  3. 使用BD GolgiStop TM (BD Biosciences,目录号:554715)的BD Cytofix/Cytoperm 固定/透化溶液试剂盒。
  4. 1×磷酸盐缓冲盐水(PBS)
  5. FACS染色缓冲液(1×PBS,2%FBS)
  6. 佛波醇12-豆蔻酸酯13-乙酸酯(PMA)(Sigma-Aldrich,目录号:79346)
  7. 离子霉素(Sigma-Aldrich,目录号:I9657)

设备

  1. BECKMAN离心机和转子(Beckman Coulter)
  2. 孵化器
  3. 96孔板
  4. Falcon圆底管

程序

  1. 刺激细胞
    1. 为了刺激从T细胞释放IFN-γ,向细胞培养物中加入PMA(5ng/ml)和离子霉素(500ng/ml),并在37℃下孵育6小时。
    2. 每6毫升细胞培养物加入4微升BD GolgiStop TM ,并彻底混合(建议BD GolgiStop不能在细胞培养中保存超过12小时)。

  2. 收获细胞
    1. 收集细胞并以1,500 rpm离心3分钟。
    2. 用PBS洗两次。
    3. 在含有2%FBS的PBS中稀释单细胞悬浮液至1×10 7个细胞/ml。
    4. 在96孔板中每孔加入100μl细胞(1×10 6个/孔)
    5. 在4℃下以800×g离心3分钟旋转板
    6. 用冷PBS洗涤3次,如步骤7一样旋转

  3. 染色细胞表面抗原
    1. 重悬细胞在100微升的Fc块(推荐稀释:1:1,000在PBS/2%FBS)。 在冰上孵育10分钟。 旋转。
    2. 重悬于100μl表面抗体混合物(推荐稀释度:1:100在PBS/2%FBS中)。 在室温下孵育20分钟,在黑暗中。 旋转。
    3. 用冷PBS洗一次。

  4. 染色细胞内抗原
    1. 重悬在200μl的BD Cytofix/CytoPerm溶液中。 在室温下孵育30分钟,在黑暗中。 Spin 1500 x g ,3分钟,4℃
    2. 用200μlBD Perm /洗涤缓冲液洗涤两次。 旋转,如步骤12.
    3. 重悬在100微升细胞因子染色(推荐稀释:1:100在1倍电极/洗涤)。 在冰上孵育,在黑暗中30分钟。 旋转,如步骤12.
    4. 用BD Perm/Wash洗涤两次,如步骤12中旋转。
    5. 重悬细胞在300-400微升FACS缓冲液,并转移到Falcon圆底管在流式细胞仪上获取。

致谢

该方案以前用于Assenmacher等人。 (1994)和Shang 等人(2004)。

参考文献

  1. Assenmacher,M.,Schmitz,J。和Radbruch,A。(1994)。 活化鼠T辅助淋巴细胞中细胞因子的流式细胞术测定:白细胞介素-10在干扰素-γ 和在白细胞介素-4表达细胞中。 Eur J Immunol 24(5):1097-1101。
  2. G,G,G,G,G,G,G,G,G,G G,G G G G G G G G G G G G G G G G G G G G G G G G 。,Simpson,AJ,Old,LJand Chen,WF(2004)。 自发的CD8 + T细胞对HLA-A2限制性 NY-ESO-1b肽在肝细胞癌患者中的作用。临床癌症研究(Clin Cancer Res)10(20):6946-6955。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, H. (2012). Intracellular Cytokine (INF-gamma) Staining Assay. Bio-protocol 2(7): e122. DOI: 10.21769/BioProtoc.122.
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Tong Gao
University of Texas
I have the same questions, when I do stimulation of mouse splenocytes with 500 ng/ml ionomycin, 500 ng/ml PMA and Golgistop at the same time fro about 4 hours, the cells looked like they all die or lyse. So I have to stop at this step. Did I add too much PMA? but according to BD protocol, IL-17 stimulation need this condition. Should I add Golgistop right after stimulation?
8/30/2016 7:50:47 AM Reply
Thankyou for this protocol. I am following almost same protocol but PMA I am using is 25-50 ng.I am not able to get intracellular stain. My intrest is CD4 cells producing IFN gamma and CD4 cells producing IL-17 A. Please guide me

Thanks
Regards

Priyanka
10/8/2012 11:26:51 AM Reply
Huagang Zhang
Yeshiva University

Hi Priyanka,
Thank you for your inquiry. Before we can get any solutions, I think it is important to find out whether the cells are not activated (ineffective stimulator?) or the staining procedure is not right (ineffective fixation/permilizaiton or bad antibodies?). So please help me answer the following questions which might help us figure out what's wrong with your experiment.
1. What type of cells are you using? Is it PBMC from human blood?
2. How long did you stimulate the cells? Is there any cell-aggregation (clusters) present in the culture?
3. Did you add BD GolgiStop right after you add PMA/Ionomycin?
4. What's the signal level of CD4 staining? There are reports stating that PMA stimulation might downregulate the expression of surface CD4, so an intracelluar CD4 staining might be helpful (http://www.ncbi.nlm.nih.gov/pubmed/11506744).
5. Did you incubate the antibodies in the 1X Perm/Wash buffer?
6. Are you able to observe any fluorescent signals in your samples?
I will get back to you as soon as I get your answers.
Thanks,
Huagang

10/9/2012 10:52:09 AM


Tong Gao
University of Texas

I have the same questions, when I do stimulation of mouse splenocytes with 500 ng/ml ionomycin, 500 ng/ml PMA and Golgistop at the same time fro about 4 hours, the cells looked like they all die or lyse. So I have to stop at this step. Did I add too much PMA? but according to BD protocol, IL-17 stimulation need this condition. Should I add Golgistop right after stimulation?

8/30/2016 7:45:04 AM