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2.4. Crystal violet staining assay
Cell survival was measured using crystal violet staining to observe the inhibition of recombinant adenovirus on the growth of the PC-3-luc cells. The PC-3-luc A 83 01 were cul-tured at 2 £ 105 cells/well in 12-well cell culture plates, and cultured at 37˚C and in 5% CO2 for 24 hours. Subse-quently, the PC-3-luc cells were infected with different concentrations of recombinant adenovirus (1 multiplicity of infection [MOI], 10 MOI, and 100 MOI). At 24, 48, 72, and 96 hours, the culture medium in each well was discarded
and the plates were turned over on a filter paper for 1 min-ute, and then 350 ml of 0.1% crystal violet solution were added to each well for the staining at room temperature for 10 minutes. The staining solution was carefully sucked dry by turning the plates over on a filter paper. The inhibitory effects of the recombinant adenovirus on the growth of the PC-3-luc cells were then observed.
PC-3-luc cells were cultured at 2 £ 105 cells/well in 12-well cell culture plates, and cultured at 37˚C and in 5% CO2 for 24 hours. Subsequently, the cells were infected with different concentrations of recombinant adenovirus (1 MOI, 10 MOI, and 100 MOI). At 24, 48, 72, and 96 hours, the PC-3-luc cells were harvested and washed 3 times with PBS. PC-3-luc cells were resuspended in 50 ml PBS and then 1 ml of the Hoechst solution was added. After 15 minutes of incubation, a 10 ml sample was applied to a microscope slide with a cover slip, and then observed and photographed under a fluorescence microscope (BX-60, Olympus, Tokyo, Japan). Tests were performed in triplicate and at least 500 cells were scored for each sample to deter-mine the nuclear changes.
2.7. Annexin V analysis
PC-3-luc cells were infected with the recombinant adenoviruses (100 MOI). After 48 hours, the cells (2 £ 105) were harvested and resuspended in binding buffer and stained with fluorescein isothiocyanate (FITC)-labeled Annexin V (Annexin V-FITC Apoptosis Detection Kit; BioVision, Mountain View, CA) according to the man-ufacturer’s protocols. To exclude late apoptotic and necrotic cells, PI was added to the FITC-Annexin V-stained samples. The samples were then examined by flow cytome-try (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ) for apoptosis analysis (Cell Quest Pro, Becton Dickinson).
2.8. Caspase analysis
The PC-3-luc cells were cultured at 2 £ 105 cells/well in 12-well cell culture plates, at 37¯C and in 5% CO2 for 24 hours. The cells were infected with 100 MOI recombi-nant adenovirus for 48 hours. The cells were then harvested and washed 3 times with PBS. PC-3-luc cells were resuspended with lysis buffer before total protein was extracted. The caspase−−3, 6, and 7 activities were then analyzed using Caspase Activity Assay Kits (Beyo-time Institute of Biotechnology, Shanghai, China). The untreated PC-3-luc cells were set as controls.
JC-1 can detect qualitative and quantitative changes in mitochondrial membrane potential (MMP).
PC-3-luc cells were cultured at 3 £ 105 cells/well in 6-well cell culture plates with a sterile cell slide placed in each well, and cultured at 37˚C and 5% CO2 for 24 hours. Subsequently, the cells were infected with recombinant adenovirus at 100 MOI. A 6-well plate was selected at 3 different time points (24, 48, and 72 hours), the liquid was discarded, 1 ml JC-1 solution was added, and the plate incu-bated for 15 minutes in the dark. The plates were washed 3 times with PBS and then the cells were observed and photo-graphed using fluorescence microscopy.
PC-3-luc cells were cultured at 1 £ 106 cells/well in 6-well cell culture plates, at 37˚C and 5% CO2 for 24 hours. When the cells became more than 90% confluent, the cul-ture medium in each well was discarded. A sterile micropi-pette tip was then used to make a scratch in the cell layer from one end of the 6-well cell culture plate to the other. The cells were washed with PBS 3 times and images were captured under an inverted microscope (0 hour). Subse-quently, the PC-3-luc cells were infected with recombinant adenovirus at 1 and 10 MOI. The 6-well cell culture plates photographed under an inverted microscope at 24 and 48 hours. The width of the scratches at each time point was measured. The experiment was repeated 3 times, and cell migration was calculated according to the following for-mula: Cell mobility = (0 hour scratch width - 24/48 hours scratch width)/0 hour scratch width.
2.11. Transwell invasion and migration assay
PC-3-luc cells were cultured at 5 £ 104 cells/well in a 24-well cell culture plate at 37˚C and 5% CO2 for 24 hours. The cells were infected with recombinant adenovirus at 10 and 100 MOI for 24 and 48 hours. The cells were then seeded in the upper chamber of the cell culture inserts after trypsiniza-tion, and cultured for 24 hours. Cells that had migrated through the membrane were counted under a microscope after they were fixated by carbinol and stained with crystal violet. The experimental procedure of matrigel invasion assay was the same as that for the transwell migration assay except for incubation with matrigel (1:7 with DMEM) in the upper chamber for 1 hour before seeding the cells.