Fig. 5

Processing of tri-cultures in special agarose molds reveals docetaxel-induced increase of apoptosis and reduction of proliferation of external melanoma cells. Tri-culture spheroids were generated by 3D cultivation of fibroblasts for 3 days, followed by simultaneous addition of keratinocytes and melanoma cells. HaCaT cells were labeled with CellTrackerRed CMPTX dye and SK-MEL-28 cells with CellTrackerGreen CMFDA dye. For the mold technique, spheroids were transferred on day five into 3D-agarose molds and then treated with 0.01 ‰ of DMSO as control or 100 nM docetaxel in DMSO for 72 h. Washing and embedding for cryosectioning occurred in the molds, as well. For the samples without mold, spheroids handled as in Figs. 3 and 4, i.e. they were treated in the cell repellent plate and then transferred to an Eppendorf tube for washing and embedding. Subsequently, all spheroids were cryosectioned into 20-μm thick slices and stained for either Ki67 or CAS3 as indicated (a). Scale bars: 100 μm. b-d Quantification of total numbers of external SK-MEL-28 cells (b), as well as amounts of Ki67- (c) and CAS3-positive external SK-MEL-28 cells (d) of tri-culture spheroids processed with or without mold. Graph displays mean ± SEM (n ≥ 3 independent experiments; ** P < 0.01). For each experiment, ≥ 3 spheroids were analyzed