The staining was performed according to the manufacturers instructions. Briefly, the cells were suspended in a buffer solution at a final concen tration of 1 106 cells ml. After addition of Annexin V FITC, the cells were incubated for 15 min at room temperature without light exposure. Flow cytometry was performed using the FACS LSR II system. A total of 10,000 www.selleckchem.com/products/Cisplatin.html ungated events were acquired for each sample, and data were analysed with the BD FACS DivaW with the CSD module. In order to determine late apoptotic cells, PI was added to the samples. Flow cytometry was performed immediately thereafter. In vivo tumourigenicity study A total of 300,000 T3M4 cells in 200 ul RPMI 1640 medium were injected subcutaneously behind the anter ior forelimb of four week old athymic mice through a 26 gauge needle.

The injection sites were examined daily for the appearance of tumours. Treatment was started on the seventh day after tumour inoculation. Mice were divided into groups receiving belinostat, gemcitabine, or a combination of both i. p, whereas the control group received only PBS. Treatment was continued for 28 consecutive days, and tumours were measured twice weekly with Vernier callipers. Tumour volumes were calculated using the formula tumour volume 2, with L representing the length and W the width of the tumour. The same treatment was performed after tumour in oculation by direct injection in the pancreatic tail via laparotomy. In this case, the tumours were compared at the end of 28 days of treatment. After completion of treatment, the animals were sacri ficed, and tumours were excised, fixed, and embedded in paraffin.

The numbers of mice in each treatment cohort was 6. All experiments on animals were approved in ac cordance with German law on the care and use of la boratory animals. Immunohistochemistry Paraffin embedded tissue sections were deparaffinised in xylene and rehydrated in progressively decreasing concentrations of ethanol. Thereafter, slides were placed in washing buffer. Antigen retrieval was car ried out by microwaving the tissue sections in 10 mM citrate buffer for 10 min. Sections were then incubated first with normal goat serum for 45 min to block non specific binding sites, and then with a mouse monoclonal Ki 67 antibody, diluted 1 5. Incubation was performed for 18 h at 4 C.

Slides were then rinsed in washing buffer and incubated with a biotinylated sec ondary goat anti mouse antibody for 45 min at room temperature. The slides were then washed in washing buffer, and each section was exposed to 100 ul DAB chromogen substrate mixture, then counterstained with Mayers haema Dacomitinib toxylin. The sections were washed again, dehydrated in increasing concentrations of ethanol, and mounted with xylene based mounting medium. Every staining was con trolled with a negative control. For semi quantitative analysis, slides were scored in a blinded manner by two observers.