Fluorescent target array killing assay: a multiplex cytotoxic T-cell assay to measure detailed T-cell antigen specificity and avidity in vivo
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Updated: May 21, 2026
The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo
Published on: June 19, 2014
Benjamin J C Quah1, Danushka K Wijesundara, Charani Ranasinghe
1Department of Immunology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, 2601, Australia. ben.quah@anu.edu.au
Abstract:
Here we describe a multiplex, fluorescence-based, in vivo cytotoxic T-cell assay using the three vital dyes carboxyfluorescein diacetate succinimidyl ester, cell trace violet, and cell proliferation dye efluor 670. When used to label cells in combination, these dyes can discriminate >200 different target cell populations in the one animal due to each target population having a unique fluorescence signature based on fluorescence intensity and the different emission wavelengths of the dyes. This allows the simultaneous measurement of the in vivo killing of target cells pulsed with numerous peptides at different concentrations and the inclusion of many replicates. This fluorescent target array killing assay can be used to measure the fine antigen specificity and avidity of polyclonal cytotoxic T-cell responses in vivo, immunological parameters that were previously impossible to monitor.
Insights
This study introduces a novel multiplex assay to track cytotoxic T-cell activity in vivo. The method uses fluorescent dyes to distinguish over 200 target cell populations, enabling detailed analysis of T-cell responses.
Area of Science:
- Immunology
- Cell Biology
- Assay Development
Background:
- Assessing cytotoxic T-cell (CTL) responses in vivo is crucial for understanding adaptive immunity.
- Existing methods often lack the multiplexing capacity to analyze fine antigen specificity and avidity simultaneously.
Purpose of the Study:
- To develop and validate a multiplex, fluorescence-based assay for in vivo cytotoxic T-cell killing.
- To enable the simultaneous measurement of antigen specificity and avidity of polyclonal CTL responses.
Main Methods:
- Utilized three vital dyes (carboxyfluorescein diacetate succinimidyl ester, Cell Trace Violet, and Cell Proliferation Dye eFluor 670) for multiplex cell labeling.
- Developed a fluorescent target array killing assay capable of discriminating over 200 distinct target cell populations within a single animal.
- Enabled simultaneous measurement of in vivo killing of target cells pulsed with various peptides at different concentrations.
Main Results:
- Demonstrated the ability to uniquely label and distinguish >200 target cell populations using a combination of fluorescent dyes based on distinct fluorescence signatures.
- Successfully measured the in vivo killing of multiple target cell populations simultaneously within the same animal.
- Facilitated the assessment of fine antigen specificity and avidity of polyclonal cytotoxic T-cell responses.
Conclusions:
- The developed fluorescent target array killing assay significantly advances the ability to study cytotoxic T-cell responses in vivo.
- This assay allows for unprecedented monitoring of immunological parameters, including fine antigen specificity and avidity, which were previously challenging to assess.
