Figure 6

Sandwich ELISA to detect secreted form of death ligands in conditioned medium obtained from MMC treated hepatoma cells. (A) Sandwich ELISA for quantification of secreted FasL from untreated and treated HepG2 and Hep3B cells at different time points. (B) Sandwich ELISA for quantification of secreted TRAIL from untreated and treated HepG2 and Hep3B cells at different time points. Data presented are representative of three independent experiments performed in triplicates expressed as mean ± s.d. *** differs significantly from the respective control. (C) Fluorescent photomicrographs of co-cultured cells to evaluate MMC induced bystander killing of HepG2-EGFP cells: (a) untreated effector HepG2 cells, (b) effector cells treated with MMC (150 nM), (c) neutralizing anti-FasL antibody (1 μg/ml), (d) neutralizing anti-TRAIL antibody (2 μg/ml). (D) Photomicrographs of co-culture of effector and target Hep3B cells: (a) untreated effector Hep3B cells with target Hep3B cells, (b) MMC (150 nM) treated effector Hep3B cells with target Hep3B cells, (c) effector Hep3B cells treated with MMC and target Hep3B cells co-plated in the presence of neutralizing anti-TRAIL antibody (2 μg/ml), (d) effector Hep3B cells pretreated with EDTA (2 μM) followed by MMC treatment and then target Hep3B cells were co-plated.