Determination of ACD. Caspase activity was measured using the Apo-ONE homogeneous Caspase-3/7-assay (Promega) according to the manufacturers protocol. Briefly, 50 000 cells/well were plated in 96-multiwell dishes, allowed to attach for 24 h, and treated with isolated compounds for 24 h. Then 50 µL of Apo-ONE Caspase-3/7-reagent was added, and the increase in fluorescence was measured for3hat 37 °C using a Perkin-Elmer Wallac VICTOR 1420 multilabel counter (excitation: 485 nm, emission: 535 nm). To investigate nuclear fragmentation as a further feature of apoptotic cell death, cells were
grown in 35 mm cell culture dishes for 48 h and then incubated with different concentrations of the flavonoid compounds for 24 h. Then, without medium change, nuclei were stained with 100 µmol/L Hoechst 33342 for 15 min and sealed with a glass coverslip. Cells with condensed and fragmented nuclei were photographed at 400-fold magnification using a Zeiss Axiolab fluorescence microscope (excitation: 365 nm, emission: 420 nm). |
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