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

  • 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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Literature Cited

Literature Cited
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