Soft-agarose colony formation assays to evaluate the role of secreted FasL in mediating cytotoxic effect. Base agarose layer was made in DMEM without phenol red and top agarose layer containing 4 × 103 HepG2 cells was poured over the base layer. After 4 weeks of growth in semisolid medium untreated and treated effector cells (5 × 105) were added in the center well of the plates. These plates were incubated at 37°C for 4 weeks. (A) Number of colonies decreased in the presence of effector cells treated with MMC as compared to control effector cells. The number of colonies decreased drastically in the presence of recombinant FasL (6 μg) which served as a positive control. (B) Photomicrographs of soft-agarose plate containing MMC treated effector HepG2 cells. The colony size diminished and very small colonies (<20 μm in mean diameter) were observed in the area surrounding center well containing treated effector cells. (C) Photomicrographs of soft-agarose colonies at 4× magnification and 10× magnification corresponding to different fields from the plate containing control effector cells and the plate containing MMC treated effector cells, in the center well.