Co-culture experiments to evaluate MMC induced bystander killing. (A) Bystander effect mediated by effector HepG2 cells treated with MMC (0.3 nM, 3 nM, 30 nM and 150 nM) for 24 h, towards target HepG2-EGFP cells. Effector cells were plated in a 12-well culture plate at a density of 5 × 104 cells per well and kept for adherence at 37°C for 24 h. MMC treatment was given for 24 h, after that medium was decanted and cells were washed twice with medium. 5 × 104 target cells were co-plated and the co-cultures were allowed to grow at 37°C for 72 h. Subsequently cells were trypsinized and total live fluorescence was quantified. In order to avoid any possibility of cell death due to residual drug in the well, we used an empty well containing no effector cells but only medium containing 150 nM MMC and subsequently this well was processed similarly before plating the target HepG2-EGFP cells. (B) Bystander effect mediated by effector Hep3B cells treated with MMC (30 nM and 150 nM) towards target HepG2-EGFP cells. (C) Bystander effect mediated by effector Hep3B cells towards target Hep3B cells. MTT assay was performed to evaluate bystander cytotoxicity in Hep3B cells. Cells were plated and treated with MMC (30 nM, 150 nM and 200 nM) for 24 h and target Hep3B cells were co-plated as described in materials and methods. After 72 h medium was decanted and 50 μl MTT (1 mg/ml) was added in each well and incubated for 4 h at 37°C. Subsequently, formazan crystals were solubilized in 50 μl of iso-propanol by incubating with shaking for 10 min, at room temperature. Absorbance was measured at 570 nm using 630 nm as reference filter. Data presented are representative of three independent experiments performed in triplicates and expressed as mean ± s.d. * and ** significantly differs from there respective controls.