Fig. 5.

Fluorescent drug accumulation in Cal33 HNC MCTSs. (A) Concentration dependence. Test drugs were added to wells containing Cal33 MCTSs, formed overnight from seeding 5,000 cells per well in ULA plates, at final concentrations of 25, 12.5, 6.25, 3.12, 1.56, or 0.781 μM and plates were incubated for 15 min before fixation in formaldehyde containing Hoechst for 30 min. After washing with PBS, DAPI, FITC, and TRITC images of Cal33 MCTSs were acquired on the IXM by using a 4 × objective and analyzed by using the MMCS image analysis module. (B) Time-dependence test compounds were added to wells containing MCTSs at a final concentration of 10 μM and incubated for 2.5, 5, 10, 20, and 30 min. Assay plates were then fixed and stained with Hoechst for 30 min, and after washing with PBS were imaged and analyzed on the IXM HCS platform by using the MWCS image analysis module. The mean ± SD (n = 3) of the mean integrated fluorescent intensity values from triplicate wells for each compound concentration (A) and timepoint (B) are presented: ellipticine-FITC (●), idarubicin-FITC (○), idarubicin-TRITC (▲), doxorubicin-TRITC (■), and daunorubicin-TRITC (□). Representative data from one of three independent experiments are shown.