Quantification of Th1 and Th17 Cells with Intracellular Staining Following PMA/Ionomycin Stimulation

Fridrik Karlsson1, Mina Hassan‐Zahraee2

1 CTI‐Boston, Pfizer Inc, Boston, Massachusetts, 2 Biotherapeutics Clinical R&D, Precision Medicine, Pfizer Inc, Cambridge, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.35
DOI:  10.1002/0471142956.cy0635s71
Online Posting Date:  January, 2015
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Abstract

Cytokine‐producing cells are at the center of the adaptive immune responses, and quantifying these cells is an important aspect to build understanding of the immune response. In particular, Th1 and Th17 cells have been implicated in the pathogenesis of such diseases as inflammatory bowel disease, rheumatoid arthritis, and multiple sclerosis. Quantification of Th1 and Th17 cells can provide important information in research of these diseases and other Th1‐ and Th17‐mediated immune disorders. In vitro stimulation of cells followed by surface and intracellular staining, presented here, has the advantage of detecting the cytokines directly instead of relying exclusively on surrogate surface markers which, although showing enrichment for the effector T cells, are not specific markers for the cytokine‐producing cells. © 2015 by John Wiley & Sons, Inc.

Keywords: Th1 cells; Th17 cells; intracellular staining; flow cytometry; adaptive immunity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Quantification of Th1 and Th17 Cells with Intracellular Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Quantification of Th1 and Th17 Cells with Intracellular Staining

  Materials
  • Ficoll‐Paque Premium (GE Healthcare Life Sciences, cat. no. 17‐5442‐02)
  • SepMate‐50 tubes (Stemcell Technologies, cat. no. 15450)
  • Human blood
  • PBS ( appendix 2A)
  • PBS with 2% FBS (P2F)
  • AO/PI staining solution (Nexcelom, cat. no. CS2‐0106‐5ML)
  • Activation medium (see recipe)
  • Fixable viability dye eFluor 506 (eBioscience, cat. no. 65‐0866)
  • FcR blocking reagent (Miltenyi Biotec, cat. no. 130‐059‐901)
  • Anti‐human CD3, Brilliant Violet 785 (Biolegend, cat. no. 317329)
  • Anti‐human CD4, Brilliant Violet 421 (BD, cat. no. 562424)
  • Anti‐human CD8, Qdot 705 (Life Technologies, cat. no. Q10059)
  • Fixation/permeabilization solution (eBioscience, cat. no. 00‐5521‐00)
  • Permeabilization buffer (eBioscience, cat. no. 00‐8333)
  • Anti‐human IL‐17A, Alexa Fluor 488 (eBioscience, cat. no. 53‐7179‐41)
  • Anti‐human IFN‐γ, PE‐CF594 (BD Horizon, cat. no. 562392)
  • 15‐ml and 50‐ml conical tubes
  • 0.5‐ml microcentrifuge tubes
  • Cellometer counting chamber (Nexcelom, cat. no. CHT4‐PD100)
  • Cellometer Auto 2000 cell viability counter (Nexcelom)
  • 96‐well round bottom plates
  • Incubator
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Figures

Videos

Literature Cited

Literature Cited
  Cosmi, L., Maggi, L., Santarlasci, V., Liotta, F., and Annunziato, F. 2014. T helper cells plasticity in inflammation. Cytometry A 85:36‐42.
  Eastaff‐Leung, N., Mabarrack, N., Barbour, A., Cummins, A., and Barry, S. 2010. Foxp3 +regulatory T cells, Th17 effector cells, and cytokine environment in inflammatory bowel disease. J. Clin. Immunol. 30:80‐89.
  Kallas, E.G., Gibbons, D.C., Soucier, H., Fitzgerald, T., Treanor, J.J., and Evans, T.G. 1999. Detection of intracellular antigen‐specific cytokines in human T cell populations. J. Infect. Dis. 179:1124‐1131.
  Kemp, K. and Bruunsgaard, H. 2001. Identification of IFN‐gamma‐producing CD4+ T cells following PMA stimulation. J. Interferon Cytokine Res. 21:503‐506.
  Mahnke, Y.D., Beddall, M.H., and Roederer, M. 2013. OMIP‐017: Human CD4(+) helper T cell subsets including follicular helper cells. Cytometry A 83:439‐440.
  Miossec, P. and Kolls, J.K. 2012. Targeting IL‐17 and TH17 cells in chronic inflammation. Nat. Rev. Drug Discov. 11:763‐776.
  Monteleone, I., Sarra, M., Pallone, F., and Monteleone, G. 2012. Th17‐related cytokines in inflammatory bowel diseases: friends or foes? Curr. Mol. Med. 12:592‐597.
  Pelchen‐Matthews, A., Parsons, I.J., and Marsh, M. 1993. Phorbol ester‐induced downregulation of CD4 is a multistep process involving dissociation from p56lck, increased association with clathrin‐coated pits, and altered endosomal sorting. J. Exp. Med. 178:1209‐1222.
  Petermann, F. and Korn, T. 2011. Cytokines and effector T cell subsets causing autoimmune CNS disease. FEBS Lett. 585:3747‐3757.
  Roederer, M., Brenchley, J.M., Betts, M.R., and De Rosa, S.C. 2004. Flow cytometric analysis of vaccine responses: How many colors are enough? Clin. Immunol. 110:199‐205.
  Weyand, C.M., Goronzy, J., and Fathman, C.G. 1987. Modulation of CD4 by antigenic activation. J. Immunol. 138:1351‐1354.
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