Measurement of Cytokine Production Using Whole Blood

Cary W. Thurm1, John F. Halsey1

1 IBT Reference Laboratory, ProGene Biomedical, Lenexa, Kansas
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.18B
DOI:  10.1002/0471142735.im0718bs66
Online Posting Date:  May, 2005
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Whole blood (WB) ex vivo stimulation assays are useful for measuring cytokine responses due to the easy access of samples from healthy donors and patients and the minimal processing of the sample required. Because the assay mimics the natural environment, WB culture may be the best milieu in which to study cell activation and cytokine production in vitro. Whole blood stimulation has been used to investigate the cellular responsiveness to a variety of stimuli, including bacterial endotoxin (LPS), antigens, allergens, and antibiotics. Various clinical uses of whole blood stimulation assays have been suggested, including the assessment of autoimmune diseases, the monitoring of drug and vaccine efficacy, and immunotoxicity. Thus, whole blood cell culture may be useful in studying the biological effects of potential allergenic and/or antigenic substances or drugs on immune cell activation and cytokine secretion.

Keywords: whole blood ex vivo stimulation; antigen; mitogen; cytokine; innate immunity; cell‐mediated immunity

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Table of Contents

  • Basic Protocol 1: Ex Vivo Stimulation of Whole Blood
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Ex Vivo Stimulation of Whole Blood

  • Antigen or mitogen (see Table 7.18.1)
  • Inhibitors (e.g., dexamethasone)
  • RPMI 1640 medium supplemented with 2 mM L‐glutamine, room temperature
  • Whole blood sample collected into sodium heparin anti‐coagulant (typically 15 USP sodium heparin/ml of blood)
  • Polypropylene 1.5‐ml microcentrifuge tubes or 24‐well Costar polystyrene tissue culture plates, or 5‐ml 12 × 75–mm Falcon polypropylene round‐bottom culture tubes
  • 37°C, 5% CO 2 humidified incubator
    Table 7.8.1   MaterialsAntigen/Mitogen Concentrations

    Antigen Final concentration Incubation time Cytokines analyzed
    LPS 4–50 ng/ml 4–8 hr TNF‐α, IL‐1β, IL‐6, IL‐8
    PHA 0.5–10 µg/ml 4–24 hr TNF‐α, IL‐1β, IL‐6, IL‐8, IL‐2, IL‐4, IFN‐γ, IL‐10
    Candida albicans soluble extract 40 µg/ml 4–24 hr TNF‐α, IL‐1β, IL‐2, IL‐6, GM‐CSF, IFN‐γ
    Tetanus toxoid 0.1–1.0 Lf/ml 2–48 hr TNF‐α, IL‐1β, IL‐2, IL‐6, GM‐CSF, IFN‐γ
    CpG DNA 10 µM 4–8 hr TNF‐α, IL‐1β, IL‐6, IL‐8
    Zymosan 100–500 ng/ml 4–24 hr TNF‐α, IL‐1β, IL‐6, IL‐8
    Anti‐CD3 10 µg/ml 4–24 hr TNF‐α, IFN‐γ, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10

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Literature Cited

Literature Cited
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