Undifferentiated cells are most susceptible to butyrate induced apoptosis, and that is associated with their poor k-calorie burning of butyrate. Under the conditions employed, Caco 2 cells were vunerable to butyrate induced apoptosis, but the on-set of cell death was not observed until 48 hmuch slower than was observed with TNF a and butyrate co incubation. In this paper, the features of TNF a/butyrate induced apoptosis of CaCo 2 cells, are defined, and the capability of certain caspase inhibitors Icotinib to prevent the cell death observed is discussed. Z AEVD. fmk and Z IETD. fmk were received from R&D Systems and kept as 20 mM stock solutions in DMSO, at _20jC until use. Anti caspase 10 IgG, anti caspase 8 IgG and anti active caspase3 were obtained from R&D Systems. Anticaspase3 IgG was received from Santa Cruz Biotechnology. Biotinylated goat anti rabbit IgG and Avidin D Texas Red were received from Vector Laboratories. Human recombinant TNF a obtained from Chemicon International and stored in aliquots of 0. 1 mg/ml at _20jC until use. Salt butyrate was obtained from Sigma and prepared as a M solution in sterile water and stored at _20jC until use. For routine passage, the human colorectal adenocarcinoma cell line, CaCo 2, was maintained in DMEM supplemented with 10% FCS, glutamax, 4. 5 g/l glucose, 2 mM sodium pyruvate, non essential amino acids, 0. 2-5 U/ml rh insulin, 100 Ag/ml streptomycin and 100 U/ml penicillin. All media items Cholangiocarcinoma were obtained from Invitrogen. Structure culture pockets were from Corning and Orange Scientific. For fluorescence microscopy centered assays, cells were seeded onto etched glass coverslips in six well plates, at a density of 2 _ 105 cells/well in 2 ml of medium. For cell proliferation assays, cells were seeded at 5 _ 103 cells/well in 100 Al of method, in 96 well plates. For circulation cytometric assays, cells were seeded at 5 frazee 105 cells/ flask in 5 ml of medium, in 25 cm2 flasks. For-all forms, cells were treated 72 h after plating. Before therapy, the cell culture medium was changed into a two weeks serum containing Capecitabine price medium, which was otherwise similar in every other aspects to the usual maintenance medium Six well culture dishes containing cells grown on coverslips were aspirated and the cells set by addition of 2 ml of pre chilled acetone/methanol at _20jC. Cells were set for 3 min and then air dry for 1 h, before storage at _20jC before staining. For discoloration, coverslips were taken off the freezer and allowed to come to room temperature before immersion in 4V,6Vdiamidino2 phenylindole solution for 3 min. DAPI solution was prepared fresh from a 5 mg/ml stock in methanol, stored at _20jC. Before mounting on glass slides using Vectorshield anti fade mount, coverslips were then rinsed 3 times in PBS.