We have created several different T cell lines specific for tumor-associated antigens, and now have developed and validated a cell-mediated cytotoxicity assay to see if the tumor antigens are presented by tumor cells to the cytotoxic T cells.


Using the IncuCyte® S3 to Image Co-Cultures and Observe Cell Killing

  • In this assay, we used two breast cancer cell lines: AU565 and MCF7
  • We labeled both cell types with IncuCyte® Cytolight Rapid Red Dye for easy identification
  • We added the following materials to our 96-well plates:
    • Antigenic peptide to half of the wells to serve as a positive control
    • HER2-specific T cells (369–377) to all wells 
    • IncuCyte® Caspase-3/7 Green Reagent to all wells, which fluoresces green when cleaved by caspase
  • We collected images of the co-cultures every hour for 18 hours using the IncuCyte® S3 Live-Cell Analysis System



We used the presence of overlapping red and green fluorescence to indicate tumor cells that had been attacked by Her2 specific T cells. Using this method, we observed cytotoxicity with both tumor cell targets (AU565 and MCF7) in the absence of the antigenic peptide, suggesting that the peptide presented by the tumor cell was produced endogenously. The amount of tumor cell death increased with increasing numbers of T cells.

We detected greater cytotoxicity of AU565 targets than MCF7 targets, which makes sense given AU565 is known to express higher levels of HER2 than MCF7.




The wide arrow points to a tumor cell under attack by two T cells. T cells are not stained with Cytolight Red and are significantly smaller than the tumor cells. The yellow-green color indicates activation of Caspase.


We also ran a similar experiment using HPV E7-specific T cells (11–20) and the CaSki cervical carcinoma cell lines as a target cell. CaSki cells express HLA0A*02:01 and HPV E7, but do not appear to be killed by the HPV E7-specific T cells. When the antigenic peptide, E7 11-20, was added there was tumor cell lysis. This suggests that the CaSki tumor cells do not present the necessary peptide on the cell surface to enable cytotoxicity.




These T cell-mediated cytotoxicity experiments demonstrate the importance of establishing which epitopes of tumor antigens are relevant for therapeutic success. Our antigen-specific T cells are critical tools for expanding our knowledge of T cell functions for the development and advancement of new immunotherapies.

Utilizing the IncuCyte® S3, we expect to apply advanced live-cell imaging techniques as a tool for understanding the dynamics of cell killing, cell migration, and phagocytosis to bring you even more advanced immune cell products and contract research capabilities.

Browse our available inventory of exclusive antigen-specific T cells or learn more about our research service offering throughout the drug development and cell therapy lifecycle.


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