Cell viability and cytotoxicity assays measure cellular changes associated with cell death, such as loss of membrane integrity, modifications in protein expression, and in enzymatic activity upon exposure to a test compound. Cell viability assays or testing are also repeatedly used over a period of time to investigate the cell proliferation within a given cell population. In cell-mediated cytotoxicity assays, the viability of target cells interacting with immune effector cells, like T, NK cells, and macrophages, is measured upon treatment with immunomodulators. Cell-mediated cytotoxicity is indeed crucial to investigate the mechanism of action and the potency of novel therapeutics and is often complemented by the characterization of effector cells' phenotype and cytokine secretion detection.
The carboxyfluorescein diacetate succinimidyl ester (CFSE) is a cell-permeable and non-fluorescent amine-reactive dye, which in viable cells is hydrolyzed by esterases in a fluorescent ester retained by the cell. The fluorescent intracellular labeling of live cells provides a powerful tool to monitor generations of proliferating cells.
In this study, the CFSE assay was used to monitor T cell proliferation in a regulatory T cell suppression assay to evaluate the immunomodulatory potency of novel test compounds. The resulting CFSE proliferating cell percentages allowed for a clear understanding on the action of the test compounds.
The antibody-dependent cellular cytotoxicity (ADCC) assay can be used to assess different mechanisms of actions of therapeutic antibodies. During the ADCC, the antibody of interest bridges the target cell, which is bound specifically via the variable region with the NK cell, which binds via its CD16 receptors to the Fc part of the antibody. Crosslinking of CD16 results in NK cell activation, degranulation, and target cell killing. Read-out of this assay is therefore the cytotoxic activity of the effector cells.
In this study, different concentrations of Rituximab were used to investigate the dose-dependent effect of this therapeutic antibody via an ADCC assay. When CD56-positive cells were removed from the sample, the remaining NKT cells do not affect the ADCC assay as NK cells kill target cells to the same extent.
All of the tools you need to perform crucial cellular cytotoxicity assays. Tune in as we share insights into isolation and expansion of your NK cells, through to full flow cytometry phenotypic characterization and analysis.
The capacity of cytotoxic T cells to specifically recognize and kill target tumor cells upon immune stimulation can be assessed via co-culture, through a T cell killing assay. T cell killing assays are widely used to investigate novel anti-cancer immunotherapies, such as a CAR T cell-based therapy.
In this study by Schäfer et al. 2021 (Nature Communications), killing assays were used to validate CD66c and TSPAN8 as target candidates for chimeric antigen receptor (CAR) T cell based immunotherapy of pancreatic adenocarcinoma. CAR T cells were successfully activated and recognition of specific target candidates was proven effective.