The effect of doxorubicin (DOX) and cisplatin (CIS) on cell proliferation. (A) Effects of doxorubicin and cisplatin on cell proliferation in multicellular structures and cell monolayers. After 48 hours treatment of cells with anticancer agents, BrdU was added to the cultures for 5 hours. Cells were trypsinised, fixed, permeabilised, exposed to anti-BrdU antibody, stained with anti-mouse conjugated to Alexa™ 488 nm and analysed with FACS. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. The data are presented as mean ± SEM from at least four separate experiments. (B) Effects on staining patterns of proliferating cell nuclear antigen (PCNA) by doxorubicin and cisplatin. Frozen sections of multicellular structures were cut, fixed and stained with anti-PCNA antibody. Ishikawa, RL95-2 and KLE 3D multicellular structures were studied. PCNA is stained green and the nucleus is blue. The scale bar is 50 μm. The images were representative of at least two separate experiments. (C) Fluorescent intensity profile of PCNA of control and DOX treated Ishikawa spheroids.