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Molecular & Genomic Testing
HIV Reservoir & Virology
Immunology
Sample Processing & Biobanking
Therapeutic Modalities
Short-term effector assays such as IFN-γ ELISpot and intracellular cytokine staining (ICS) measure what a responding cell does within hours of antigen stimulation. However, an effective antigen-specific T cell response also depends on clonal expansion, and measuring the proliferative capacity and phenotype of responding cells is critical to fully resolving the quality of this response. On its own, our T cell proliferation assay identifies which antigen-specific T cells in a sample retain the capacity to activate and expand, independent of their ability to produce a given cytokine. Run alongside ELISpot and intracellular cytokine staining on the same sample, these assays deliver a functional profile that neither effector frequency nor proliferation describes alone.
To quantify antigen-induced T cell proliferation ex vivo, we use flow cytometry to follow CFSE dye dilution as responding T cells expand. Loaded prior to antigenic stimulation, the CFSE dye covalently labels intracellular proteins and is partitioned approximately equally between daughter cells at each division. Successive generations therefore resolve as discrete peaks of halving fluorescence, and the profile records division history at the single-cell level. Co-staining for lineage, phenotype, and activation markers attributes that history to defined cell subsets. Because a small number of antigen-specific precursors can give rise to many labeled progeny over several days of culture, this assay may also uncover responses too infrequent to resolve in other assays.
Stable intracellular dye
Multi-day antigen exposure
Subset and phenotype markers
Read on a flow cytometer
Cells, typically peripheral blood mononuclear cells (PBMCs), are labeled with carboxyfluorescein succinimidyl ester (CFSE), a fluorescent dye that binds stably inside the cell.
Labeled cells are exposed to the antigen, peptide pool or mitogen of interest, with appropriate control conditions, and cultured over a multi-day period.
After culture, cells are stained with antibodies that identify the CD4+ or CD8+ T cell subsets of interest (e.g., memory markers) and their phenotypic state (e.g., activation and/or exhaustion markers).
Cells are read on a flow cytometer. Each round of division halves the dye intensity, so dividing cells appear as a series of dimmer populations compared with the undivided cells. Results are reported against the control conditions for each sample, so the response to a specific stimulus can be separated from background division.
Proliferation sits alongside ELISpot, ICS, cytokine profiling and cytolytic function assays in the same functional immunology program, so the method can be matched to the question.
Programs can start in the vivarium of your choice and continue into analytically qualified testing for clinical trials, without changing partners along the way.
Molecular detection, immune phenotyping and functional assays can be run from the same sample, which keeps results comparable across endpoints.
One team of scientists stays with your program from protocol design to final report, and the people who design the assay are the people who review the data.
Because the dye is tracked cell by cell, the assay reports division history rather than a single bulk number. That makes it possible to see which subsets expanded and how the responding fraction compares across conditions. It also supports low-frequency responses, because dividing cells accumulate over the culture time.
Cell viability matters in any proliferation readout. Dead cells can take up dyes and antibodies nonspecifically, so a viability marker is included at staining and dead cells are excluded before the proliferating cells are analyzed.
Applying the same gating approach across every sample and condition, in a study design established during scoping, helps build confidence that differences between conditions reflect cell growth rather than differences in sample quality. The presence of a dividing population is then read against the control conditions, in order, sample by sample.
Each T cell assay looks at a different part of the response. Proliferation adds something the others do not: it shows whether antigen-specific cells can expand. For cell therapy, that connects to a practical question, which is whether the product is expanding, persisting and functioning. For gene therapy, proliferative recall can be read next to anti-capsid antibody and T cell responses. In both settings, phenotype, persistence and functional potency together give a fuller picture than any single readout.
| Assay | what it measures | ||
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ELISpot
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Counts cells that secrete a cytokine after short stimulation, reported as spot-forming units per million cells
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ICS |
Identifies which cell subsets produce which cytokines, using up to about ten markers in a standard panel
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Deep profiling
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Extends phenotyping and functional readouts to panels of 10 to 20 markers
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CFSE dye dilution
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Measures proliferative capacity, with up to two markers in the assay
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Our T cell proliferation assay services are designed around your sample type, your stimulus and the decision the data will support. A cell proliferation assay can be built on more than one chemistry, and the right choice depends on the question. For programs that need more than one measurement of the same response, cell proliferation assays can be run together with other functional assays in the same study, using the same sample where the design allows.
The default for T cell work because it reports division history and can be combined with subset identification and activation markers.
Report DNA synthesis across a cell population. They show that cells in the culture are capable of dividing, but they do not identify which subsets divided.
Such as CellTiter-Glo report the overall metabolism of a culture. They are simpler, but they give a population-level signal and cannot tell you which cells divided. They are better suited to bulk cell growth questions than to antigen-specific T cell work.
Each program is scoped to the study. Deliverables are agreed in the statement of work and are designed to be decision-ready, with consistent, longitudinal data from the first sample to the final follow-up visit. Qualification documentation is available to support regulatory filings where the program requires it.
Cell therapy. Proliferation and functional readouts help characterize whether an engineered or expanded cell product is expanding, persisting and functioning. Studies commonly pair proliferation with phenotyping, so that the identity of the dividing cells is known.
Gene therapy. T cell proliferation can be used alongside anti-capsid antibody and T cell response assays to characterize immune responses to a vector or its payload.
Immunology research programs. Proliferation is used to characterize antigen-specific responses in preclinical and clinical studies, including low-level responses that may be missed by short-term secretion assays.
Accelevir supports research use. Results are provided to inform study decisions and are not intended for diagnostic purposes.
when the question is whether antigen-specific cells can expand, or when you expect a low-level response that benefits from a longer culture.
when you need to know which subsets produce which cytokines, and how those functions co-occur in the same cell.
when you need a sensitive count of cytokine-secreting cells. Our ELISpot assay services include single-color assays reported as spot-forming units per million cells, and FluoroSpot for multi-analyte readouts.
For a side-by-side scientific comparison of CFSE and ICS, see CFSE T cell proliferation assay vs intracellular cytokine staining. Together, these methods form an immune response profiling strategy. Our immune response profiling page describes how ICS, ELISpot and cytokine profiling are combined, and our cytokine multiplex page covers secreted-protein readouts. If your question is about target cell elimination rather than expansion, a T cell killing assay measures cytotoxic function directly. Our cytolytic function assays page describes the cytotoxicity assay formats we run. For cell identity and marker panels, see our flow cytometry services and phenotyping.
Understanding the limits of a method is part of using it well.
What it captures
Whether cells in a sample divided in response to a stimulus, compared with control conditions.
Which subsets divided, when the panel includes subset markers.
Low-level responses that accumulate over a culture period.
Proliferation shows that cells divided. It does not show what those cells secrete or whether they can kill a target. Those questions need cytokine and cytolytic assays.
Sample quality also matters. Cryopreservation and handling affect how cells respond, so we recommend discussing sample collection and storage during scoping. See our page on cryopreserved PBMCs for more.
A T cell proliferation assay measures whether T cells divide in vitro after exposure to an antigen, peptide pool or mitogen. Because expansion depends on cells being able to respond and multiply, the readout complements assays that measure cytokine secretion or killing.
CFSE is a fluorescent dye that labels cells. Each time a cell divides, the dye is shared between the daughter cells, so fluorescence intensity halves. Flow cytometry reads the resulting series of dimmer populations as division history.
A proliferation assay measures whether cells expand. A T cell killing assay measures whether effector cells eliminate target cells, and a cytotoxicity assay measures cell death. They answer different questions and can be paired in the same program.
Thymidine incorporation and BrdU report DNA synthesis across a population, and ATP-based assays report the metabolic activity of the culture. CFSE dye dilution tracks division cell by cell and can be combined with subset markers. The right choice depends on the question, and for antigen-specific T cell work CFSE is generally the more informative option.
A viability marker is included at staining so dead cells can be excluded before proliferating cells are analyzed. This helps keep nonspecific staining from being mistaken for a response.
Yes. For cell therapy, proliferation and functional readouts help address whether a product is expanding, persisting and functioning. For gene therapy, T cell proliferation can be studied alongside anti-capsid antibody and T cell responses.
Start with the question. CFSE dye dilution addresses whether cells can expand, ICS identifies which subsets produce which cytokines, and ELISpot counts cytokine-secreting cells. Many programs combine them. Our scientists can recommend a combination during scoping.
Often, yes. Molecular detection, immune phenotyping and functional assays can be run from the same sample, and the study design during scoping determines what is feasible for your material.
Scientific scoping, a tailored proposal, a statement of work with e-signature, and delivery of decision-ready data, all handled by one scientist-run team.
Yes. Accelevir operates as a CAP-accredited, CLIA-certified laboratory.
Tell us about your sample, stimulus and the question you need answered, and our scientists will scope the approach with you.
