EPA-FFA was a gift from SLA Pharma AG (Watford, UK), and is referred to henceforth as EPA. Use of EPA in in vitro experiments has been described previously [12 (link)]. PGE2, PGE1 alcohol and ONO-AE3-208 were obtained from Cayman (USA). PD98058 was obtained from Sigma (UK).
MC38 mouse CRC cells were a gift from D. Beauchamp (Vanderbilt University, TN, USA). HCA-7 human CRC cells were obtained from Sigma-Aldrich and RAW264.7 mouse macrophages were obtained from the ATCC (www.atcc.org ). HCA-7 cells were authenticated by STR profiling. Cells were cultured in RPMI 1640 medium containing Glutamax® supplemented with 10% (v/v) FBS, at 37°C in a humidified atmosphere containing 5% CO2.
Cell chemosensitivity to EPA was determined using the MTT assay [30 (link)]. Briefly, cells were seeded in 96-well plates and incubated overnight at 37°C. The following day, the medium was removed and replaced with fresh medium containing EPA for 96 hrs at 37°C. Cell survival was determined using the MTT assay. IC50 values were calculated using Prism6™ software and are expressed as the mean ± standard deviation of three independent experiments.
Culture medium was conditioned by 1×105 cells in 6-well plate wells at 37°C for 24 hrs, in the presence or absence of 50-200μM EPA, EP receptor agonists/antagonist and 30μM PD98058. CCL2 levels were measured using a mouse or human CCL2 immunoassay (both R&D Systems).
MC38 mouse CRC cells were a gift from D. Beauchamp (Vanderbilt University, TN, USA). HCA-7 human CRC cells were obtained from Sigma-Aldrich and RAW264.7 mouse macrophages were obtained from the ATCC (
Cell chemosensitivity to EPA was determined using the MTT assay [30 (link)]. Briefly, cells were seeded in 96-well plates and incubated overnight at 37°C. The following day, the medium was removed and replaced with fresh medium containing EPA for 96 hrs at 37°C. Cell survival was determined using the MTT assay. IC50 values were calculated using Prism6™ software and are expressed as the mean ± standard deviation of three independent experiments.
Culture medium was conditioned by 1×105 cells in 6-well plate wells at 37°C for 24 hrs, in the presence or absence of 50-200μM EPA, EP receptor agonists/antagonist and 30μM PD98058. CCL2 levels were measured using a mouse or human CCL2 immunoassay (both R&D Systems).