Proliferation assay - 3H thymidine incorporation

Analyzing lymphocyte proliferation in response to allogeneic, antigen-specific or polyclonal stimulation can be a useful tool to determine the immunomodulatory effects of biologics or other drugs.

Total PBMCs can be used as a starting population to keep the assay as similar to the in vivo situation as possible. Alternatively, a co-culture of (monocyte-derived) dendritic cells and T cells can be set up to specifically screen successful presentation of immunogenic peptides to T cells, characterized by increased proliferation of the latter population.

Also see our other proliferation assay by flow cytometry.

Examples of proliferation assessment via 3H thymidine incorporation

Comparison of the effect of 3 biosimilars on lymphocyte proliferation after an MLR

Here, the effect of low and high doses of 3 different biosimilars (A, B and C) on lymphocyte proliferation have been tested. PBMCs from two healthy donors have been co-cultured in an MLR. Lymphocyte proliferation was measured by the high-troughput method of 3H thymidine incorporation.

Assessing lymphocyte proliferation after dendritic cell immunogenic peptide presentation to T cells

The T cell response to immunogenic peptides presented by dendritic cells (DC) was assessed using 3H thymidine incorporation as a read-out. After loading the DCs with the peptides, T cells were added to the culture. Proliferation during 24 hours was measured after 7 days of co-culture. A no peptide control was included to correct for basal T cell proliferation in this set up. A response to the presented peptide was defined as values >3 SD above the no peptide control.

For more applications, please see the references below.


Guidelines for analysis of low-frequency antigen-specific T cell results: Dye-based proliferation assay vs 3 H-thymidine incorporation
Di Blasi D, Claessen I, Turksma AW, van Beek J, ten Brinke A.
J Immunol Methods 2020;487:112907. doi: 10.1016/j.jim.2020.112907.

Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses
ten Brinke A, Marek-Trzonkowska N, Mansilla MJ, Turksma AW, Piekarska K, Iwaszkiewicz-Grześ D, Passerini L, Locafaro G, Puñet-Ortiz J, van Ham SM, Hernandez-Fuentes MP, Martínez-Cáceres EM, Gregori S.
Front Immunol 2017;8:1870. doi: 10.3389/fimmu.2017.01870.


Need help?

Get in touch with Anja ten Brinke, PhD or Annelies Turksma, PhD
[email protected]