1%, triton X-100 0.1% and propidium iodide 50 μg/ml) (Nicoletti et al., 1991), and the cell fluorescence was determined by flow cytometry, as described above. The mitochondrial transmembrane potential was determined by the retention of rhodamine 123 dye (Gorman et al., 1997 and Sureda et al., 1997). The cells were washed click here with PBS, incubated with rhodamine 123 (5 μg/ml, Sigma Chemical Co. St Louis, MO, USA) at 37 °C for 15 min in the dark and washed twice. The cells were then incubated again in PBS at 37 °C for 30 min in the dark and their fluorescence was measured
by flow cytometry, as described above. Phosphatidylserine externalisation was analysed by flow cytometry (Vermes et al., 1995). A Guava® Nexin Assay Kit (Guava Technologies, Hayward, CA) determined LY2835219 chemical structure which cells were apoptotic (early apoptotic + late apoptotic). The cells were washed twice with cold PBS and then re-suspended in 135 μl of PBS with
5 μl of 7-amino-actinomycin D (7-AAD) and 10 μl of Annexin V–PE. The cells were gently vortexed and incubated for 20 min at room temperature (20–25 °C) in the dark. Afterwards, the cells were analysed by flow cytometry, as described above. Caspase 3/7 activity was analysed by flow cytometry using the Guava® EasyCyte Caspase 3/7 Kit (Guava Technologies, Hayward, CA). The cells were incubated with Fluorescent Labelled Inhibitor of Caspases (FLICATM) and maintained for 1 h at 37 °C in a CO2 incubator. After incubation, 80 μl of wash buffer was added and the cells were centrifuged at 2000 rpm for 5 min. The resulting pellet was resuspended in 200 μl of wash buffer and centrifuged. The cells were then re-suspended in the working solution (propidium iodide and wash buffer) and analysed immediately using flow cytometry, as described above. The drop test assay determined the relative sensitivity of different S. cerevisiae strains to ATZD treatment. The following S. cerevisiae strains were used: BY-4741, Top1Δ and Top3Δ. Cells were treated with ATZD mafosfamide at concentrations of 50 and 100 μg/ml and more, 4
dilutions 1:10 were performed. A suspension of 2 × 105 cells/ml of S. cerevisiae in the exponential phase was used. An aliquot of 3 μl of each dilution was added to plates containing YEPD medium (YEL + agar). After 3–4 days of growth at 28 °C, the plates were photographed. m-AMSA served as the positive control. The inhibitory effects of ATZD on human DNA topoisomerase I were measured using a Topo I Drug Screening Kit (TopoGEN, Inc.). Supercoiled (Form I) plasmid DNA (250 ng) was incubated with human Topo I (4 units) at 37 °C for 30 min in relaxation buffer (10 mM Tris buffer pH 7.9, 1 mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine and 5% glycerol) in the presence or absence of ATZD (50 and 100 μg/ml, final 20 μl). The concentrations used were based on the positive control indicated in this Kit. CPT (100 μM) served as the positive control.