Related Experiment Video
Updated: Aug 9, 2026
Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
Published on: November 9, 2017
Enumeration of cytokine-secreting cells at the single-cell level
B J Skidmore1, S A Stamnes, K Townsend
1Lilly Research Laboratories, La Jolla, CA 92037.
Insights
A new enzyme-linked immunosorbent assay (ELISA) method accurately quantifies single cells producing interferon-gamma (IFN-gamma) or tumor necrosis factor (TNF). This sensitive technique allows for the measurement of cytokine-secreting cells in various immune cell populations.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Quantifying cytokine secretion at the single-cell level is crucial for understanding immune responses.
- Existing methods may lack sensitivity or require large cell numbers.
Purpose of the Study:
- To develop a sensitive and reliable assay for the quantitation of single cells secreting interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF).
- To demonstrate the applicability of this method across different cell types and stimulation conditions.
Main Methods:
- Development of an enzyme-linked immunosorbent assay (ELISA) using antibody-coated microwells.
- Stimulation of cloned T cells and cells from lymphoid organs, bone marrow, and peripheral blood.
- Detection of cytokine secretion via enzyme-labeled antibody conjugates and substrate, forming visible spots.
Main Results:
- Successfully quantitated single cells secreting IFN-gamma from stimulated T cells and lymphoid organs.
- Detected and quantitated TNF-secreting cells from normal lymphoid tissues, bone marrow, and peripheral blood.
- Demonstrated the assay's sensitivity and potential for broad cytokine detection.
Conclusions:
- The developed ELISA is a sensitive method for quantifying single cytokine-secreting cells.
- This technique is adaptable for measuring the frequency of cells secreting various cytokines, provided specific antibodies are available.
- Offers a valuable tool for immunological research and diagnostics.
Abstract:
A sensitive assay utilizing enzyme-linked immunosorbent assay methodology has been developed for the quantitation of single cells secreting interferon (IFN)-gamma or tumor necrosis factor (TNF). Cloned T cells or cells from lymphoid organs were stimulated with antigen, concanavalin A, or phorbol myristate acetate and ionomycin in microwells coated with antibodies specific for IFN-gamma. Discrete "spots" overlying areas where cells secrete IFN-gamma were then developed by incubation with a second antibody to IFN-gamma, followed by an enzyme-labeled antibody conjugate and substrate. Similarly, using TNF-specific antibody reagents, TNF-secreting cells were detected and quantitated in cell populations obtained from normal lymphoid tissues, bone marrow and peripheral blood, following activation with phorbol myristate acetate and ionomycin. Provided specific antibodies are available, this method has the potential to measure the frequency of cells secreting any cytokine.
