NFkB-luciferase reporter assay

The in-house engineered Jurkat/hOX40 cells, were stably transfected to generate a stable cell line that co-expressed an NFkB-luciferase reporter plasmid, pGL4.10[luc2] (Promega). Bright-Glo luciferase reagent and a luminometer were used to assess activation of NFkB signaling. The expression vector, luciferase reagent, and luminometer (Promega) were used according to manufacturer guidelines. Briefly, anti-CD3 (OKT3, at 1 μg/mL) was diluted in PBS and used to coat white 96-well plates (Costar) over night at 4 °C. The next morning, plates were washed to remove unbound anti-CD3 and 10,000 Jurkat/hOX40/NFkB-luciferase cells were plated in each well with 2 μg/mL functional grade CD28 (eBiosciences), +/− the ARC or other test agents. Plates were incubated at 37 °C/5% CO₂ for 6 h, where after the Bright-Glo reagent was added and luminescence was assessed on a luminometer.