Flow Cytometric Analysis of Cytokine Responses in Stimulated Whole Blood: Simultaneous Quantitation of TNF‐α‐Secreting Cells and Soluble Cytokines

Arancha Rodríguez‐Caballero1, Andrés García‐Montero1, Clara Bueno1, Alberto Orfao1

1 Universidad de Salamanca, Salamanca, Spain
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.21
DOI:  10.1002/0471142956.cy0921s25
Online Posting Date:  August, 2003
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Abstract

This unit describes technical protocols aimed at the ex vivo or in vitro evaluation of the functional status of the immune system through the simultaneous identification and enumeration of cytokine‐secreting cells and quantitation of the soluble cytokines produced by these cells.

Keywords: Cytokines; TNF‐α; immune system; cell activation; immunoassay

     
 
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Table of Contents

  • Basic Protocol 1: Simultaneous Detection of Cell Activation and Quantitation of the Secreted Cytokines
  • Alternate Protocol 1: Measurement of Generic Inflammatory Response Induced by Lipopolysaccharide
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Simultaneous Detection of Cell Activation and Quantitation of the Secreted Cytokines

  Materials
  • Human peripheral whole blood
  • Vacutainer tubes containing heparin (Becton Dickinson)
  • Cell culture medium (see recipe)
  • 1 µg/ml phorbol myristate acetate (PMA; see recipe)
  • 50 µg/ml ionomycin stock solution (see recipe)
  • 1% (v/v) DMSO in PBS ( appendix 2A for PBS)
  • 100% ethanol
  • 2 mM TACE inhibitor (see recipe)
  • Antibody‐coated bead solutions: coated with anti‐IFN‐γ, anti‐TNF‐α, anti‐IL‐2, anti‐IL‐4, anti‐IL‐5, and anti‐IL‐10 MAbs (e.g., BD Biosciences)
  • Fluorochrome‐labeled monoclonal antibodies:
    • Anti human TNF‐α‐PE (e.g., Mab 11, BD Biosciences)
    • Anti human CD45‐PE‐Cy5 (e.g., J33 MAb, Immunotech)
    • Anti human CD3‐FITC (e.g., HIT3a, BD Biosciences)
    • Anti human CD8‐APC (e.g., B9.11, Immunotech)
    • Mix of anti‐cytokine (IFN‐γ, TNF‐α, IL‐2, IL‐4, IL‐5, and IL‐10) PE‐conjugated MAbs (e.g., BD‐CBA kit, BD Biosciences)
  • Recombinant IFN‐γ, TNF‐α, IL‐2, IL‐4, IL‐5, IL‐10 cytokine standards (see recipe) and appropriate assay diluent (e.g., BD Biosciences)
  • PBS ( appendix 2A)
  • Flow cytometer calibration beads (unstained, FITC‐, PE‐, PE‐Cy5‐, and APC‐stained BD CaliBRITE beads, BD Biosciences) and cytometer setup beads (including FITC‐ and PE‐positive control detectors; BD‐CBA kit, BD Biosciences)
  • 12 × 75–mm round‐bottomed polystyrene tubes (Falcon, BD)
  • Humidified 37°C, 5% CO 2 cell incubator
  • Flow cytometer equipped with two lasers (emitting at 488 nm and at 630 nm) and with a filter set for detection of fluorescence emission at 530 ± 30, 585 ± 42, 661 ± 16, and ≥670 nm and appropriate software for instrument setup, data acquisition, and analysis
  • Spreadsheet database software (e.g., Microsoft Excel)
NOTE: It is important that all volumetric measurements be very precise. See unit 6.4 for reverse pipetting technique.

Alternate Protocol 1: Measurement of Generic Inflammatory Response Induced by Lipopolysaccharide

  • 2.5 µg/ml bacterial endotoxin lipopolysaccharide (LPS; see recipe)
  • Beads coated with anti‐TNF‐α, anti‐IL‐1β, anti‐IL‐6, anti‐IL‐8, anti‐IL‐10, and anti‐IL‐12 MAbs (e.g., BD Biosciences)
  • Fluorochrome‐labeled monoclonal antibodies:
    • Anti human CD14‐FITC (e.g., MφP9, BD Biosciences)
    • Mix of anti‐cytokine (TNF‐α, IL‐1β, IL‐6, IL‐8, IL‐10, and IL‐12) PE‐conjugated MAbs (e.g., BD‐CB kit, BD Biosciences)
  • Recombinant TNF‐α, IL‐1β, IL‐6, IL‐8, IL‐10, and IL‐12 cytokine standards (e.g., BD Biosciences; see recipe)
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Figures

Videos

Literature Cited

   Arai, K.I., Lee, F., Miyajimza, A., Miyatake, S., Arai, N., and Yokota, T. 1990. Cytokines: Coordinators of immune and inflammatory responses. Annu. Rev. Biochem. 59:783‐836.
   Bueno, C., Almeida, J., Alguero, M.C., Sanchez, M.L., Vaquero, J.M., Laso, F.J., San Miguel, J.F., Escribano, L., and Orfao, A. 2001. Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: Comparative analysis of different stimuli, secretion‐blocking agents and incubation periods. Cytometry 46:33‐40.
   Cook, E.B., Stahl, J.L., Lowe, L., Chen, R., Morgan, E., Wilson, J., Varro, R., Chan, A., Graziano, F.M., and Barney, N.P. 2001. Simultaneous measurement of six cytokines in a single sample of human tears using microparticle‐based flow cytometry: Allergics vs. non‐allergics. J. Immunol. Methods 254:109‐118.
   Manz, R., Assenmacher, M., Pflüger, E., Miltenyi, S., and Radbruch, A. 1995. Analysis and sorting of live cells according to secreted molecules, relocated to a cell‐surface affinity matrix. Proc. Natl. Acad. Sci. U.S.A. 92:1921‐1925.
   Mascher, B., Schlenke, P., and Seyfarth, M. 1999. Expression and kinetics of cytokines determined by intracellular staining using flow cytometry. J. Immunol. Methods 223:115‐121.
   Prussin, C. and Metcalfe, D.D. 1995. Detection of intracytoplasmic cytokines using flow cytometry and directly conjugated anticytokine antibodies. J. Immunol. Methods 188:117‐128.
Key References
   Bueno, C., Rodriguez‐Caballero, A., García‐Montero, A., Pandiella, A., Almeida, J., and Orfao, A. 2002. A new method for detecting TNF‐α‐secreting cells using direct‐immunofluorescence surface membrane staining. J. Immunol. Methods 264:77‐87.
  First description of the method of detection of TNF‐α on the membrane of the secreting cells, showing its sensitivity compared to the intra‐cytoplasmatic detection.
   Chen, R., Lowe, L., Wilson, J.D., Crowther, E., Tzeggai, K., Bishop, J.E., and Varro, R. 1999. Simultaneous quantitation of six human cytokines in a single sample using microparticle‐based flow cytometric technology. Clin. Chem. 45:1693‐1694.
  Short report in which authors describe the bead‐based immunoassay and its specificity, sensitivity, and range compared with a similar conventional ELISA assay.
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