Fig. 2. Enhancement of TNF-R1-induced cell death by costimulation of TNF-R2 occurs in various cell lines. (A) Jurkat cells stably transfected with TNF-R2 were seeded in 96-well microtiter plates at a density of 50x10³ cells/well and were treated overnight in triplicates with TNF, crosslinked FasL or crosslinked TRAIL with ([UNK]) or without () costimulation of TNF-R2. (B) Colo205 cells were seeded in 96-well microtiter plates at a density of 10x10³ cells/well and cultured overnight at 37°C. The next day the cells were treated for 48 hours in triplicates with TNF or crosslinked TRAIL with ([UNK]) or without () costimulation of TNF-R2. (C) Kym1 cells were seeded in 96-well microtiter plates at a density of 15x10³ cells/well and cultured overnight at 37°C. The next day the cells were treated overnight in triplicates with the TNF-R1-specific agonistic mAb Htr1 with () or without () costimulation of TNF-R2. As stimulation of TNF-R2 results in Kym1 cells in significant induction of endogenous TNF (Grell et al., 1999), the action of endogenous TNF was suppressed by addition of TNF-R1-Fc (10 µg/ml) and TNF-R1 triggering was aquired by the agonistic mAb Htr1c. Viable cells were quantified in all experiments by the MTT method.