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Updated: Aug 8, 2026
Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
Published on: November 9, 2017
Single-cell cytokine profiles in normal humans: comparison of flow cytometric reagents and stimulation protocols
1Immunology Branch, Centers for Disease, Control and Prevention (CDC), U.S. Department of Health and Human Services (DHHS), U.S. Public Health Service (PHS), Atlanta, GA 30333, USA.
Insights
Intracellular cytokine analysis in normal adults reveals significant inter-individual variability. Flow cytometry demonstrated diverse T-cell cytokine expression patterns, highlighting method limitations and reagent utility.
Area of Science:
- Immunology
- Cellular Biology
Background:
- Cytokines are crucial signaling molecules functioning at the microenvironmental level.
- Intracellular cytokine assessment has become increasingly feasible with advanced techniques.
- Understanding cytokine profiles in normal individuals is essential for interpreting immune responses.
Purpose of the Study:
- To evaluate the applicability and comparability of different directly conjugated anti-human cytokine reagents.
- To assess various stimulation protocols for intracellular cytokine detection in peripheral blood lymphocytes.
- To characterize cytokine production patterns in T lymphocytes from healthy adults.
Main Methods:
- Utilized 3-color flow cytometry to analyze surface and cytoplasmic antigens.
- Examined peripheral blood lymphocytes from 18 normal donors.
- Compared results from separated cells versus whole blood preparations using PHA/PMA/ionomycin stimulation.
Main Results:
- Inter-donor variability in cytokine production greatly exceeded technique-related variability.
- Post-stimulation, T lymphocytes produced gamma-interferon (IFN) (13.2-65.0%), interleukin-2 (IL-2) (10.0-56.7%), and tumor necrosis factor-alpha (TNF-α) (17.1-79.2%).
- CD4+ T cells produced more IL-2, while CD8+ T cells produced more gamma-IFN; TNF-α expression was similar.
Conclusions:
- Normal adult T lymphocytes exhibit a broad spectrum of cytokine production patterns.
- Current cytokine detection reagents and methods have both utility and limitations.
- Significant inter-individual differences in cytokine expression should be considered in immunological studies.
Abstract:
Cytokines are produced and function at a micro environmental level: intracellular assessment has only recently become practically feasible. We used 3-color flow cytometry to examine surface and cytoplasmic antigens on peripheral blood lymphocytes of 18 normal donors, assessing the applicability/comparability of various directly conjugated anti-human cytokine reagents and stimulation protocols using separated cells or whole blood preparations. Interdonor variability far exceeded variability due to reagent or stimulation and separation techniques. Based on all results with various reagents, post 4-5.5 h stimulation with PHA/PMA/ionomycin, the range of the percents of T lymphocytes producing various cytokines included: gamma-IFN-13.2-65.0%, IL-2-10.0-56.7%, and TNF-alpha-17.1-79.2%. Compared to CD8+ cells, CD4+ cells more often expressed IL-2 (mean 45.7% of CD4 + vs. 21.4% of CD8+ p < 0.0001), less often expressed gamma-IFN (18.5% vs. 55.3%, p < 0.0001), and did not differ in TNF-alpha expression (52.9% vs. 59.4%). Of T cells producing gamma-IFN, 64.8-100.0% also produced TNF-alpha 3.5-100.0%, IL-2. Of T cells producing IL-2, 6.0-63.9% also produced gamma-IFN and 37.6-100.0%, TNF-alpha. These results demonstrate the broad spectrum of cytokine patterns in normal human adults, as well as the usefulness and limitations of various currently available cytokine products.
