Welcome guest, Sign in

Home

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Intracellular Cytokine (INF-gamma) Staining Assay

Immunology > Immune cell staining > Immunodetection
Author: Huagang Zhang
Huagang ZhangAffiliation: Albert Einstein College of Medicine, Yeshiva University, New York City, USA
For correspondence: huagangzhang@gmail.com
Bio-protocol author page: a21
Vol 2, Iss 7, 4/5/2012, 16255 views, 2 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.122

[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.


How to cite this protocol: Zhang, H. (2012). Intracellular Cytokine (INF-gamma) Staining Assay. Bio-protocol 2(7): e122. DOI: 10.21769/BioProtoc.122; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
8/30/2016 7:50:47 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?

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

10/8/2012 11:26:51 AM  

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

10/9/2012 10:52:09 AM  

Huagang Zhang (Author)
Albert Einstein College of Medicine,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

8/30/2016 7:45:04 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?

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register