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Related Experiment Video

Updated: Dec 23, 2025

Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
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Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System.

Sarah E Mann1, Zhicheng Zhou2, Laurie G Landry1

  • 1Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, Aurora, CO, United States.

Frontiers in Immunology
|April 25, 2020
PubMed
Summary

This study developed a novel multiplex assay to efficiently test antigen reactivity of multiple T cell receptors (TCRs). The system uses NFAT-based fluorescent reporters for sensitive and specific antigen discovery, reducing cost and labor.

Keywords:
T cell receptorsantigensepitopesmultiplex assayreporter

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Last Updated: Dec 23, 2025

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biotechnology

Background:

  • Single-cell sequencing identifies numerous T cell receptors (TCRs) on tumor-specific and autoimmune T cells.
  • Determining the antigen specificity of these T cells is crucial for therapeutic development.

Purpose of the Study:

  • To develop an efficient, cost-effective, and labor-saving method for testing the antigen reactivity of multiple TCRs simultaneously.
  • To enable high-throughput antigen discovery for T cell-mediated immune responses.

Main Methods:

  • Utilized a Nuclear Factor of Activated T cells (NFAT)-based fluorescent reporter system.
  • Generated T-hybridoma cell lines expressing unique TCRs and distinct fluorescent proteins for multiplexing.
  • Combined multiple T-hybridoma lines in single reactions to assess antigen stimulation.

Main Results:

  • The multiplex assay demonstrated sensitivity to antigen stimulation without decreased performance.
  • Multiplexed reactions maintained specificity, with responses from one cell line not affecting others.
  • The system allows for the simultaneous testing of numerous T cell receptors against various antigens.

Conclusions:

  • The developed multiplex assay system is a valuable tool for antigen discovery research.
  • This method facilitates the identification of T cell epitopes using techniques like combinatorial peptide libraries.
  • The assay offers a scalable and efficient approach for understanding T cell responses in diverse immunological contexts.