T cell killing assessment

Explicyte is offering a specialized in vitro T cell cytotoxicity-mediated killing assay to evaluate the ability of test compounds to modulate the immune response of effector cell subsets. Our sensitive and quantitative assay is based on a 96-w plate co-culture of human tumor cells* and primary immune cells and consists in the kinetic monitoring of tumor cell proliferation and death thereby enabling to measure the killing immune response towards tumor cells by analyzing cell death specifically within the tumor cell population.
 
* Contact us to know the tumor cell lines available in this assay
 

Illustrative data 

Immune cell killing of H1299 lung cancer cells
Immune cell-mediated killing of H1299 lung cancer cells in the presence of aCD3-activated PBMCs. H1299 tumor cells stably expressing a nuclear red fluorescent probe are cultured, at an appropriate effector:target ratio, with inactivated or aCD3-activated hPBMC. Live cell real time detection images of apoptotic H1299 tumor cells at 72h post-culture with inactivated and aCD3-activated hPBMC. Tumor cell count (nuclear red probe-expressing cells, (a, b, d, e)) and apoptosis (caspase 3/7 green fluorescent probe, (a, b, d, e)) are kinetically monitored by live cell imaging. Segmentation masks & image analysis are performed (c, f) to quantify apoptosis specifically within the tumor cell population (i.e. caspase 3/7 green fluorescent probe within the nuclear red probe-expressing cells – yellow objects). Arrows in e show apoptotic tumor cells and arrowheads apoptotic non-tumor cells.

Immune cell-mediated killing of H1299 lung cancer cells in the presence of aCD3-activated ...

Immune cell killing of A375 melanoma cells
CD8 T cell-mediated killing of A375 melanoma cells is promoted by increasing anti-CD3 concentrations. 
Melanoma A375 tumor cells were seeded and 48h later were co-cultured with activated CD8 cells, in the presence and absence of increasing concentrations of anti-CD3 antibody. (A) Tumor cell apoptosis was monitored and quantified over a period of ~3 days, by mean of a caspase 3/7 fluorescent probe and a NucRed probe expression, respectively, as surrogate measures of immune cell killing activity towards tumor cells. (B) IFN gamma released levels in the co-culture supernatants were quantified by mean of a specific HTRF detection kit. Data are expressed as means ± SEM.

CD8 T cell-mediated killing of A375 melanoma cells is promoted by increasing anti-CD3 concentrations. ...

Immune cell killing of SKOV-3 ovarian cancer cells
Real-time live cell monitoring of tumor cell killing mediated by activated PBMCs, under untreated and Atezolizumab-treated conditions. Ovarian SKOV3 tumor cells were seeded and 24h later were co-cultured with activated PBMCs, in the presence and absence of aCD3/aCD28, in the presence and absence of Atezolizumab or isotype control. Tumor cell apoptosis (A) and count (B) were monitored and quantified over a period of ~5 days, by mean of a caspase 3/7 fluorescent probe and a NucRed probe expression, respectively, as surrogate measures of immune cell killing activity towards tumor cells. Data were normalized and corrected to the baseline and are expressed as means ± SEM.

Real-time live cell monitoring of tumor cell killing mediated by activated PBMCs, under untreated ...

Assay principle

T cell killing

  • Co-culture of target tumor cells with immune cells (PBMCs, purified T cells…) in the presence of an activator cocktail to promote killing activity.
  • Real-time monitoring of tumor cell count (tumor cells stably expressing a nuclear fluorescent probe) as a surrogate of cell proliferation, and of tumor cell death using a mix-and-read caspase 3/7 fluorescent reagent, over several days in the continuous presence of immune cells.
  • Automated image analysis & segmentation enables apoptotic death quantitation specifically within tumor cells (caspase 3/7 fluorescent probe within the nuclear probe-expressing cells).
  • Ability to multiplex with additional functional measures such as effector cytokine release.