The assay was carried out in 24 well plates. Cells were treated selleck catalog with fungal taxol or fungal baccatin III for 6, 12, 24 and 36 h. The cells were then incubated with 2. 5 ug ml 1 of JC 1 dye for 15 min at 37 C, washed once with ice cold PBS containing 2% FBS, resuspended in the same and analyzed immediately by flow cytometry. JC 1 monomers emit at 530 nm and J aggregates emit at 590 nm. 2, 4 Dinitrophenol is used as the positive control to set the gates along with the untreated cells as the negative control. The percentage of MMP was plotted against time upon fungal taxol or baccatin III treatment. Data analysis was carried out using CellQuest Pro software. Determination of nuclear morphology The changes in chromatin organization upon treatment with fungal taxol or baccatin III was determined microscopically by staining either with Hoechst 33258 or acridine orange ethidium bromide dual stain.

After overnight adherence on cover slips, the cells were incubated with fungal taxol or baccatin III for 12 h. The cells were then fixed with 3. 7% paraformaldehyde, permeabilized with 0. 1% Triton X 100 and stained with Hoechst 33258. After washing twice with PBS, cells were examined by fluorescence microscopy. The apoptotic cells were identified by the presence of highly condensed chromatin or fragmen ted nuclei. For AO/EB staining, after treatment with in dicated concentrations of taxol or baccatin III for 12 h, the cells were incubated with 3 ul of RNase A at 37 C for 30 min. After washing twice with PBS, the cells were fixed with 3. 7% paraformaldehyde for 10 min at room temperature.

Then the cells were stained with an AO/ EB mixture for 15 min and washed with PBS, the cells were observed under fluorescence microscope at 10�� magnifica tion using 485 nm excitation and 535 nm emission filter sets. DNA fragmentation analysis DNA fragmentation was studied as described earlier. Jurkat cells were treated with fungal taxol or baccatin III, whereas HeLa cells, after overnight adherence were treated with fungal taxol or bacca tin III, for 36 h. After treatment, the cells were har vested and washed with 1 ml of PBS, resuspended in 100 ul of PBS and fixed in 70% chilled ethanol overnight. The cells were spun down at 1000 g and resuspended in 40 ul of phosphate citrate buffer consisting of 192 parts of 0. 2 M Na2HPO4 and 8 parts of 0. 1 M of citric acid, at RT for 30 min.

After centrifugation at 1000 g at RT for 5 min, the supernatant was transferred to fresh tubes and concentrated by vacuum in SpeedVac concentra tor. 3 ul of 0. 25% Nonidet 40 in distilled water was then added to the tubes, followed by 3 ul of a so lution Batimastat of RNase A. After incuba tion for 30 min at 37 C, 3 ul of a proteinase K was added and incubated for additional 30 min at 37 C. Gel loading buffer was the added and the entire content of the tube was transferred to 1. 2% agar ose gel and electrophoresed at 2 V/cm for 16 h.