Medium transfer experiments to detect the presence of soluble cytotoxic effector molecules. (A) Time-course release of cytotoxic factor from MMC treated effector HepG2 cells. The effector cells were treated with MMC for 24 h and fresh medium was added as described in materials and methods. At the time indicated, corresponding supernatants were collected and added to the untreated target HepG2-EGFP cells with or without neutralizing anti-FasL antibody. Target cells were incubated with the supernatants for 48 h before quantitating fluorescence. The supernatants were supplemented with 0.2% FBS to avoid cell death due to growth factor depletion. The culture supernatant from untreated HepG2 cells was used as control and fluorescent intensity was calculated with respect to identical time point controls. (B) The target cell death increases with the increase in number of effector cells. Effector cells were treated with MMC for 24 h and fresh medium was added as described in materials and methods. The supernatants were collected after 48 h and used to culture target HepG2-EGFP cells for 48 h before quantitating fluorescence. (C) Involvement of death ligands in bystander killing of hepatoma cells. Bystander killing of HepG2-EGFP cells is mediated by FasL. Effector HepG2 and Hep3B cells were treated with MMC (150 nM) and target HepG2-EGFP cells were co-plated with or without neutralizing antibody against FasL (1 μg/ml) and TRAIL (2 μg/ml). (D) Bystander killing of target Hep3B cells by effector Hep3B cells treated with MMC (150 nM) is mediated by TRAIL. Trypan blue assay to detect the percentage cell viability in co-cultures of effector and target Hep3B cells in the presence of neutralizing anti-TRAIL antibody. After 72 h of growth in co-culture cells were harvested by trypsinization and stained with 0.005% trypan blue for 5 min at RT. Unstained as well as stained cells were counted in haemocytometer and percentage cell viability was calculated. Data presented are representative of three independent experiments performed in triplicates and expressed as mean ± s.d. *, ** and *** differs significantly at p < 0.05.