Prostaglandin analysis and cAMP detection by enzyme immunoassay Competitive enzyme immunoassay was used http://www.selleckchem.com/products/z-vad-fmk.html to determine PGE2 levels in culture media in 96 well format by Assay Designs. Prostaglandin concentration was calculated using the optical density of the samples in relation to a standard curve generated by dilutions of a standard provided. Quantitative determina tion of cAMP concentration Inhibitors,Modulators,Libraries in cell lysates was performed using the cAMP immunoassay by R and D Sys Inhibitors,Modulators,Libraries tems. Adherent cells were lysed in 0. 1 N HCl for 10 min at 37 C and supernatants were assayed according to manufacturers instructions in a 96 well plate. cAMP concentrations were calculated using a similar standard curve method. Cell proliferation Cell proliferation assays were carried out using the BrdU cell proliferation ELISA according to manufacturers instructions.

Cells were seeded at 5 103well in 96 well plates and following overnight serum starvation were treated with vehicle or drug. Amphiregulin neutralisation was with anti human neutralising AR antibody from Stratech Scientific. Cell cycle analysis HT 29 cells were seeded at a density of 1 106well in 6 well plates and treated with drug or vehicle for 24 hours. Adherent cells were Inhibitors,Modulators,Libraries suspended using trypsin EDTA for 3 5 min at 37 C and were fixed overnight in 75% ethanol at 4 C then washed and resuspended in a solution of PBS containing 0. 1% Triton X 100, 0. 05 mgml of DNase free RNase and a 50 ?gml propidium iodide in the dark for 30 min. Cells were resuspended Inhibitors,Modulators,Libraries in PBS prior to analysis in a FACScalibur flow cytometer with measurement of fluorescence emission at 575 nm.

Analysis of cell cycle distribution was per formed using CellQuest. Tumour collection The protocol was approved by the Ethics Committee of Beaumont Hospital, Dublin and all patients provided written, informed consent. Samples of colorectal tumournormal were obtained Inhibitors,Modulators,Libraries from patients at the time of surgery and were immediately placed in RNAlater solution or fixed in 10% formalin. Statistical analysis A one way analysis of variance was used to examine overall differences between multiple groups, with Bonferroni multiple comparisons test. Two tailed paired students T test was used to compare the means of paired samples. The statistical packages GraphPad InStat and GraphPad Prism were used. Statistical significance was set at a P value of less than 0.

05, whereas a P value less than 0. 005 was considered highly significant. Results EP receptor expression in HT 29 cells is similar to in vivo expression in CRC EP receptor expression was assessed by qRT PCR in the human colon cancer cell line HT 29 and EP4 receptor was the most abundant receptor subtype. A Calcitriol IL-2 rep resentative amplification plot is shown in Figure 1a with linear amplification of EP4 seen in identical template after the shortest number of cycles.