Resulting DNA distributions were analysed by the CellQUEST software (Becton Dickinson, Mountain View, CA, USA) for the proportion of cells in sub-G0, G1, S, and G2�CM phases of the cell cycle. In a second series http://www.selleckchem.com/products/crenolanib-cp-868596.html of experiments, cells were treated with TNF�� (625Uml?1) alone at H24 and then cultured for 3 days. Medium was then harvested and replaced by RPMI. Cells were stained at different time points up to 21 days and analysed for DNA content on a FACScan as described above. In vivo model All the in vivo experiments were performed in compliance with the French guidelines for experimental animal studies (Agreement No. “type”:”entrez-protein”,”attrs”:”text”:”A34220″,”term_id”:”321026″A34220) and fulfil the UKCCCR Guidelines for the welfare of animals in experimental neoplasia.

Mice Athymic 7�C9-week-old female Swiss nude mice (nu/nu, Iffa Credo, l’Arbresle, France) were housed in self-contained filter-top cages (five mice cage?1) in a facility controlled for temperature, humidity, and a 12:12h light:dark cycle under sterile conditions. The animals were given autoclaved food and water ad libitum. Experimental protocols The human pancreatic carcinoma BxPC-3 cells were harvested with 0.25% trypsin solution, washed, and adjusted to 2 �� 106 150��l?1 RPMI-1640 medium without fetal calf serum. Each mouse was injected s.c. in the right flank with 150��l of the cell suspension. After 35 days, the mice were grouped according to tumour size by measuring tumour diameters with a Vernier caliper to avoid nonhomogeneous groups before beginning treatments.

Tumour dimensions were measured twice weekly and volumes (mm3) were estimated by the formula d1 �� d2 �� d3/2, where d1 is the length, d2 is the width, and d3 is the height of the tumour. On day 35, the mice were assigned to seven different treatment groups (five mice per group) Batimastat as follows: Group 1: 0.9% NaCl i.v. injection alone (200��l injection?1) for this control group on days 34, 37, 41, 44, and 48. Group 2: TNF�� at 1��g i.v.?1 injection alone (in 200��l 0.9% NaCl injection?1) on days 34, 37, 41, 44, and 48. Group 3: BAb at 25��g i.v.?1 injection alone (in 200��l 0.9% NaCl injection?1) on days 33, 36, 40, 43, and 47. Group 4: BAb+TNF�� (ratio 25��g:1��g; molar ratio 12.5:1) i.v. injection (in 200��l 0.9% NaCl injection?1) on days 33, 36, 40, 43, and 47. BAb�CTNF�� mixture was prepared 24h before injection. Group 5: Local radiation as described above delivered on days 34, 37, 41, 44, and 48+0.9% NaCl i.v. injection (200��l injection?1) 3h before irradiation. Group 6: Local radiation as described above delivered on days 34, 37, 41, 44, and 48+TNF�� i.v.