Cell viability, apoptosis and distribution of doxorubicin (DOX). (A) Cell viability after 48 hours treatment with doxorubicin (DOX) and cisplatin (CIS). The treated cells were stained with PI and analysed with FACS. The PI positive cell staining was calculated relatively to the control. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers, respectively. (B) After 48 hours treated with anticancer agents, cells were stained with annexin-FITC and PI and analysed with FACS. Multicellular structures and cell monolayers of Ishikawa, RL95-2 and KLE cells were studied. The data are presented as mean ± SEM from at least four separate experiments. The (■) dark and (grey square symbol) grey bars represent 3D multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. (■) P < 0.05, comparison of between 3D cell cultures and cell monolayers. (C) Distribution of doxorubicin (DOX) within 3D multicellular structures of Ishikawa, RL95-2 and KLE after 48 hr incubations. DOX was located in the nucleus of cancer cells in multicellular structures. Images were obtained and representative of two independent experiments.