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Updated: Jul 1, 2026
Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester
Published on: June 4, 2019
Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution
Paul K Wallace1, Joseph D Tario, Jan L Fisher
1Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. pkw@RPCIflow.org
Insights
Cell proliferation assays using dyes like CFSE track cell division for immune response studies. This method quantizes precursor cell frequency and expansion, aiding in understanding antigen-specific T-cell responses.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Cell-tracking dyes such as CFSE and PKH26 are vital for monitoring cell proliferation in flow cytometry.
- These dyes enable quantitative analysis of cell division in heterogeneous cell populations responding to immune stimuli.
Purpose of the Study:
- To detail the methodology for calculating precursor cell frequencies and expansion extent using cell-tracking dyes.
- To demonstrate the combined analysis of cell phenotype, antigen binding, cytokine production, and proliferation.
Main Methods:
- Utilizing fluorescent cell-tracking dyes (CFSE, PKH26) for quantitative proliferation analysis.
- Employing multiparameter flow cytometry to assess antigen-specific tetramer binding, cytokine production (IFN-gamma), and cell division (dye dilution).
Main Results:
- Established a method to back-calculate the number of proliferating cells from dye intensity.
- Demonstrated that only about half of influenza tetramer-binding cells proliferated and produced IFN-gamma.
Conclusions:
- Cell-tracking dyes combined with multiparameter flow cytometry offer a robust approach for characterizing complex antigen-driven immunological responses.
- Future advancements in dyes and flow cytometry technology will further enhance the analysis of cellular dynamics in immunology.
Abstract:
Cell-tracking reagents such as the green-fluorescent protein labeling dye CFSE and the red-fluorescent lipophilic membrane dye PKH26 are commonly used to monitor cell proliferation by flow cytometry in heterogeneous cell populations responding to immune stimuli. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division. Because daughter cell fluorescence intensities are approximately halved after each division, the intensity of a cell relative to its intensity at the time of staining provides information about how many divisions it has undergone. Knowing how many rounds of division have occurred and the relative number of cells in each daughter generation, one can back-calculate the number of cells in the original population (i.e., cells present at the time of stimulus) that went on to respond by proliferating. Using this information, the precursor cell frequencies and extent of expansion to a specific antigen or mitogen of interest can be calculated. Concurrently, the phenotype of the cells can be determined, as well as their ability to bind antigen or synthesize cytokines, providing more detailed characterization of all cells responding to the antigen, not just effector cells. In multiparameter flow cytometric experiments to simultaneously analyze antigen-specific tetramer binding, cytokine production and T-cell proliferation, we found that only approximately half of the cells that exhibited specific binding to influenza tetramer also proliferated, as measured by dye dilution, and synthesized IFNgamma in response to antigen. We expect the advent of new cell tracking dyes emitting from the violet to the near infrared combined with the increasing number of lasers and detectors on contemporary flow cytometers to further expand the usefulness of this approach to characterization of complex antigen-driven immunological responses.
