Detection of Intracellular Cytokines by Flow Cytometry

Yuzhi Yin1, Alyssa Mitson‐Salazar2, Calman Prussin1

1 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 2 Yale School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.24
DOI:  10.1002/0471142735.im0624s110
Online Posting Date:  August, 2015
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Abstract

Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single‐cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes. This capability, combined with the high throughput inherent in the instrumentation, gives ICCS an enormous advantage over other single‐cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell surface staining are also described. © 2015 by John Wiley & Sons, Inc.

Keywords: cytokines; CD4‐positive T lymphocytes; CD8‐positive T lymphocytes; immune system; epitopes; T lymphocyte; cytokine staining

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Intracellular Staining for Cytokines
  • Support Protocol 1: PMA and Ionomycin Activation of T Cells
  • Support Protocol 2: Antigen Activation of T Cells
  • Support Protocol 3: Fixation and Freezing of PBMC
  • Support Protocol 4: Cell‐Surface Staining of PBMC
  • Support Protocol 5: Demonstrating Specificity of Cytokine Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Intracellular Staining for Cytokines

  Materials
  • Cells: stimulated, Amine reactive viability dye stained, fixed, and frozen (see Support Protocols protocol 21 to protocol 43)
  • PBS‐S (see recipe)
  • PBS‐S/milk solution (see recipe)
  • Directly labeled anti‐cytokine MAbs (e.g., anti‐IFN‐γ Alexa Fluor 700 and anti‐IL‐13 PE‐Cy7) and anti‐surface marker antibodies (e.g., anti‐CD3 Qdot 605; anti‐CD4 PerCP‐Cy5.5; CD8 PE‐TexRed, and anti‐CD154 FITC)
  • PBS/BSA (see recipe)
  • 4‐ml V‐bottom tubes for staining (Sarstedt)
  • Tabletop centrifuge
  • Additional reagents and equipment for flow cytometric analysis (unit 5.4; Holmes et al., )

Support Protocol 1: PMA and Ionomycin Activation of T Cells

  Materials
  • Peripheral blood
  • Complete RPMI‐10 medium ( appendix 2A), 37°C
  • 10 mM ionomycin (Ca2+ salt) in DMSO (add 5 mg Ca+2 ionomycin per 0.67 ml DMSO; store at −20°C)
  • 200 μg/ml phorbol myristate acetate (PMA) in 100% ethanol (store at −20°C)
  • 10 mg/ml brefeldin A (BFA) in DMSO (store at −20°C)
  • 3 mg/ml DNase I (60,000 Dornase U/ml; Calbiochem) in PBS, optional
  • Amine reactive viability dye: LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) or equivalent product from other supplier
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 24‐well tissue culture plates
  • Cell scraper or disposable transfer pipets
  • Refrigerated centrifuge
  • Additional reagents and equipment for preparing peripheral blood mononuclear cells [PBMC; unit 7.1 (Fuss et al., )] and counting cells ( appendix 3A; Strober, )

Support Protocol 2: Antigen Activation of T Cells

  Materials
  • Peripheral blood
  • Complete RPMI‐10 medium ( appendix 2A), 37°C
  • Anti‐CD28
  • SEB (Toxin Technology)
  • 10 mg/ml brefeldin A (BFA) in DMSO (store at −20°C)
  • 3 mg/ml DNase I (60,000 Dornase U/ml; Calbiochem) in PBS, optional
  • 0.1 M EDTA in PBS (see appendix 2A for PBS)
  • 16 × 125‐mm round‐bottom polystyrene tissue culture tubes (Corning)
  • Refrigerated centrifuge
  • 4‐ml V‐bottom tubes
  • Additional reagents and equipment for preparing peripheral blood mononuclear cells [(PBMC; unit 7.1 (Fuss et al., )], counting cells ( appendix 3A; Strober, ), and cell viability staining ( protocol 4)
NOTE: SEB is a Select Agent; distribution, storage and handling are tightly regulated (http://www.selectagents.gov/).

Support Protocol 3: Fixation and Freezing of PBMC

  Materials
  • 4% paraformaldehyde, stored in aliquots frozen at −20°C (see recipe)
  • Activated PBMC (see protocol 2 or 2) or activated surface‐stained cells (see protocol 5)
  • PBS ( appendix 2A), 4°C
  • PBS/BSA (see recipe), 4°C
  • 10% (v/v) dimethyl sulfoxide (DMSO) in PBS (10% DMSO/PBS), 4°C. Use standard reagent grade (not cell culture grade) DMSO.
  • 4‐ml V‐bottomed tubes for staining (Sarstedt)
  • “Pincushion” test tube rack
  • 2‐ml freezing vials

Support Protocol 4: Cell‐Surface Staining of PBMC

  Materials
  • Stimulated cells to be stained (see Support Protocols 1 and/or 2)
  • PerCP/Cy5.5‐conjugated anti‐human CD4 MAb
  • PBS/BSA (see recipe), 4°C
  • PBS ( appendix 2A), 4°C
  • 4‐ml V‐bottomed tubes for staining (Sarstedt)
  • Refrigerated centrifuge
  • Additional reagents and equipment for counting cells ( appendix 3A; Strober, ) and fixation of cells (see protocol 4)
NOTE: It is important that all washes and incubation be done on ice to decrease secretion of intracellular cytokine.

Support Protocol 5: Demonstrating Specificity of Cytokine Staining

  Additional Materials (also see protocol 1Basic Protocol)
  • Unlabeled and FITC‐labeled anti‐IFN‐γMAb
  • Unlabeled and PE‐labeled anti‐IL‐4 MAb
  • Unlabeled and APC‐labeled anti‐IL‐2 MAb
  • Unlabeled irrelevant isotype‐control MAbs matching each of the anti‐cytokine Ig isotypes
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Figures

Videos

Literature Cited

Literature Cited
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