Colony formation assays

Cell proliferation upon liposome treatment was examined by two types of colony formation assays: monolayer and soft agar (41). For monolayer colony formation, cells were seeded onto 24-well plates and treated with free or liposomal drugs (100 nM, PCT; 90 nM, XR) for 7 days before fixation, crystal violet staining and imaging (G-box, Syngene, Frederick, MD). For soft agar colony formation, the cells mixed with 0.4% agar/medium were seeded onto 0.8% agar/medium support in 6-well plates and treated with the liposomes (100 nM, PCT) for 40 days with the media changed every week. Cells were fixed, stained with crystal violet and imaged with the G-Box bio-imaging system.