The biological activity of the fusion proteins was determined by their ability to stimulate the proliferation of murine cytotoxic T lymphocytes (CTLL2) (ATCC). In 96-well plates, cells (25’000-50’000 per well) were seeded in culture medium supplemented with serial dilutions of the fusion proteins. After incubation at 37°C for 72 hours, cell proliferation was determined with Cell Titer Aqueous One Solution® (Promega). Results were expressed as the percentage of cell viability compared to untreated cells.
Biological NF-κB activity assay was performed as described before (38 (link)). CTLL-2_ NF-κB reporter cells were starved in absence of IL2 for 6-9 hours. Cells were seeded in 96-well plates at 50’000 cell/well with serial dilutions of the fusion proteins. To assess luciferase production, 20μl of supernatant was transferred in an opaque 96-well plate (PerkinElmer, Optiplate 96, white) with 80μl of Coalenterazine (Carl Rath AG, 1μg/ml). Luminescence was immediately measured at 466nm and results were calculated and expressed by division with values obtained from untreated cells.
Biological NF-κB activity assay was performed as described before (38 (link)). CTLL-2_ NF-κB reporter cells were starved in absence of IL2 for 6-9 hours. Cells were seeded in 96-well plates at 50’000 cell/well with serial dilutions of the fusion proteins. To assess luciferase production, 20μl of supernatant was transferred in an opaque 96-well plate (PerkinElmer, Optiplate 96, white) with 80μl of Coalenterazine (Carl Rath AG, 1μg/ml). Luminescence was immediately measured at 466nm and results were calculated and expressed by division with values obtained from untreated cells.
