Propidium iodide staining of fixed cells was used to ascertain the number of cells with sub G1 fractional DNA content, as an estimate of apoptosis, according to a modified way of Darzynkiewicz et al.. Quickly, cells were harvested, washed 3 times in ice-cold PBS and finally resuspended in a volume of 1 ml PBS. Cells were fixed from the following addition of 3 ml of ice-cold absolute ethanol. Cells suspended in ethanol were kept at _20jC for up to 2 weeks. For investigation, cells were pelleted at 300 page1=39 g for 5 min. The supernatant was aspirated and the cell pellet resuspended in 2 ml PBS. The cells were spun again at 300 frazee g for 5 min and finally resuspended in 500 Al PBS. 2 hundred microliters of DNA extraction buffer was then added and the cells were incubated for 5 min at RT. Cells were pelleted by centrifugation and incubated for 30 min at room temperature and resuspended in 1 ml Icotinib of DNA staining option. Cells were then pelleted and resuspend in 1 ml FACS buffer. Data acquisition and flow cytometric analysis was performed employing a Becton Dickinson FACScan with Macintosh based CellQuest computer software. Ten thousand gated events were obtained for each data point. Data analysis was done using PC based, Winmidi software. The proportion of cells with sub G1 DNA content was used as an estimate of apoptosis. Dedication of apoptosis by TUNEL Terminal deoxynucleotide transferase mediated dUTPbiotin nick end labelling was performed using a TdT in situ package, using a modified protocol. Shortly, coverslips with cells were edged having an IMMedge pen, Immune system before rehydration in PBS for 10 min at room temperature. Coverslips were taken off the PBS and placed cell side down onto 50 Al of Cytonin, which have been spotted onto a microscope slide and incubated for 15 min at room temperature. Coverslips were then removed and washed twice in 2 ml of molecular biology grade water and once-in PBS. Coverslips were then placed cell side down on the microscope slide spotted with 50 Al of labelling reaction mixture and incubated for 1 h, at 37jC, in a humidified chamber. Coverslips were incubated for 5 min at room temperature and then transferred to 2 ml of stop buffer. Coverslips were then washed twice, for 2 min, in 2 ml of PBS before labelling with Avidin N Texas Red in the dark, for 30 min at room temperature. Cells were then washed in PBS, counterstained with DAPI and mounted, as previously described. Qualitative analysis of apoptosis order Dizocilpine by immunofluorescent labelling of active caspase 3 Cells were fixed and cultured, for morphological analysis of apoptosis. Cells were then incubated with 10% goat serum in PBS, before incubation with rabbit anti human active caspase 3 IgG in TBS/ 10% goat serum/0. 1 5 years tween 20. Biotinylated goat antirabbit IgG accompanied by Avidin D Texas Red was used for immunofluorescent recognition.