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Related Experiment Video

Updated: Nov 12, 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.

Laurie G Landry1, Sarah E Mann1, Amanda M Anderson1

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

Bio-Protocol
|March 18, 2021
PubMed
Summary

Identifying T-cell receptor (TCR) cognate antigens is crucial for understanding autoimmune diseases. This study presents a novel multiplexed reporter system for efficient screening of TCR-antigen interactions, advancing therapeutic and biomarker discovery.

Keywords:
AntigensEpitopesFluorescent protein markersMultiplex assayNFATReporterT-cell receptorsT-cell stimulation

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biotechnology

Background:

  • T-cell receptor (TCR) and major histocompatibility complex (MHC) interactions with antigens are central to immune responses and tolerance.
  • Disruptions in these interactions contribute to autoimmune diseases like type 1 diabetes.
  • Identifying specific T-cell epitopes is vital for developing therapeutics and biomarkers.

Purpose of the Study:

  • To develop a novel reporter system for T-cell activation.
  • To enable high-throughput screening of T-cell receptor (TCR)-antigen interactions.
  • To facilitate the identification of cognate antigens for T-cells.

Main Methods:

  • A reporter system utilizing ZsGreen-1 fluorescent protein driven by nuclear factor of activated T-cells (NFAT) signaling.
  • Flow cytometry for detecting T-cell activation.
  • Multiplexing up to eight different reporter T-cell lines simultaneously, each with a unique TCR and fluorescent identifier.

Main Results:

  • The reporter system allows for the indefinite use of T-cell lines after initial transduction.
  • The multiplexing capability enables the screening of numerous TCR-antigen interactions efficiently.
  • The system is versatile for studying various T-cell-MHC-antigen trimolecular interactions.

Conclusions:

  • This multiplexed reporter system significantly enhances the ability to screen TCR-antigen interactions.
  • It offers a practical approach for identifying antigenic epitopes relevant to autoimmune diseases.
  • The technology holds promise for advancing the development of novel immunotherapies and diagnostic biomarkers.