Anti bodies targeted against Phospho p44 42 MAPK and I��B were from Cell Signaling Technology and the antibody targeted against actin was from Sigma Aldrich. HRP conjugated secondary antibodies, Goat Anti Mouse IgG and Goat Anti Rabbit IgG were from Southern Biotech. Other chemicals used were H89 from Alexis Chemicals, GW5074 www.selleckchem.com/products/chir-99021-ct99021-hcl.html from Sigma Aldrich and Gefinib Iressa from Selleck Chemicals LLC. Cell culture The human colon adenocarcinoma cell line Caco 2 were grown in RPMI 1640 supplemented with 10% fetal calf serum, 100 uM non essential amino acids, 1 mM sodium pyruvate, and gentamicin. Cells were maintained in a humidified incubator at 37 C and 5% CO2. PCR RNA from both tissue and cells were isolated using Qiagen RNase Mini kit. Complementary DNA was synthesized with Super ScriptW ViloTM using 20 ul of reaction mixture containing 2 ug RNA.
PCR was set up using TaqManW Master Mix and TaqManW probes GPR120, GPR40 and rplp0. 50 ng tem plate was used in each reaction. Cytosolic Ca2 measurements Measurements of the cytosolic Ca2 concentration Inhibitors,Modulators,Libraries were performed in the following extracellular solution 150 mM NaCl, 5 mM KCl, 2. 4 mM CaCl2, 1. 3 mM MgCl2, 10 mM glucose and 10 mM HEPES, adjusted to pH 7. 4 by NaOH. Approximately 106 Caco 2 cells were seeded out in collagen coated glass bottom dishes 3 4 days prior to experiment. Cells were loaded with 5 uM of the fluorescent Ca2 indica tor fura 2 AM in EC for 45 min at 37 C, followed by washout of the fura 2 ester and fur ther 30 min incubation at room temperature. Then, cells were mounted on an Olympus OSP 3 system for dual ex citation fluorometry.
The excitation light was switched at 200 Hz between 360 and 380 nm using a ro tating mirror. The emitted fluorescence Inhibitors,Modulators,Libraries was recorded at 510 nm with a photomultiplier, and the measurements were restricted to single cells by a pinhole diaphragm. The ratio between Inhibitors,Modulators,Libraries emissions at the two different excitation wavelengths reflects the cytosolic Ca2 con centration i. In the present study, the relative in crease in i is used as a measure of the response to the PUFAs. Therefore, calibration in order to determine the absolute Ca2 concentrations was not performed. Cells were exposed to the different PUFAs by pressure ejection from a micropipette placed about 40 um from the cell. As negative control, cells were exposed to EC by pressure ejection using the same conditions as de scribed above.
No artefacts were observed when ejecting normal EC onto cells, and the increase in i was com pared to untreated cells. Cell stimulation and lysis Sodium salts of DHA, Inhibitors,Modulators,Libraries EPA and Inhibitors,Modulators,Libraries AA were dissolved in autoclaved deionized water and further diluted in RPMI 1640. 0. 5 106 cells were seeded per well in a 24 well plate. The day after, the cells were serum starved for selleck chemicals 24 h in RPMI 1640 supplemented with 1% FCS, and then stimulated as indicated. After stimulation, the cells were lysed in 100 ul lysis buffer per well for 30 min at 4 C.