Raw 264.7 mouse macrophages

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).

iNOS supporting activity was measured using a novel mouse macrophage bioassay recently reported by us¹⁴ . Briefly, RAW 264.7 mouse macrophages (ATCC, USA), were cultured using Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, UK) supplemented with 2mM L-glutamine (Sigma Aldrich, UK), nonessential amino acids (Invitrogen, UK) and penicillin-streptomycin (Sigma Aldrich, UK) at 5% CO₂ and 37 °C. At confluence, cells were scraped and spun at 400 relative centrifugal force for 5 minutes. 10% filtered foetal bovine serum (LabTech, UK) was included only when culturing. For the iNOS supporting bioassay, media was replaced with neat (100%) human plasma from patients before and at different times after cardiopulmonary bypass – this was added directly to RAW 264.7 cells. Cells were then stimulated to express iNOS with LPS (1 μg/ml) for 24 h. Plasma was then removed and immediately assays nitrite levels as a marker of iNOS activity. Nitrite was measured using the Griess assay as we have described previously¹⁴ .

RAW 264.7 mouse macrophages (ATCC, USA), were cultured using Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, UK) supplemented with 2mM L-glutamine (Sigma Aldrich, UK), nonessential amino acids (Invitrogen, UK) and penicillin-streptomycin (Sigma Aldrich, UK) at 5% CO₂ and 37°C. At confluence, cells were scraped and spun at 400 relative centrifugal force for 5 minutes. 10% filtered foetal bovine serum (LabTech, UK) was included only when culturing.

B6 fibroblasts were generated as previously described.^{83 (link)} Qa-1b expressing L cells were generated as previously described.^{28 (link)} NIH 3T3 (ATC#CRL-1658) and RAW 264.7 mouse macrophages (ATCC TIB-71) were purchased from ATCC. QFL hybridomas (BEko8Z cells) were established in the Shastri lab as described.^{28 (link)} B6 fibroblasts and splenocytes were cultured in complete RPMI (cRPMI) with 10% FBS (Invitrogen, Carlsbad CA), 100 U/ml Penicillin/Streptomycin (Invitrogen), 2mM L-Glutamine, and 50 μM 2-ME. NIH 3T3 and RAW 264.7 cells were cultured in complete DMEM with 10% FBS (Invitrogen, Carlsbad CA) and 100 U/ml Penicillin/Streptomycin (Invitrogen). All cell lines were maintained at 37°C, 5% CO₂, and 95% humidity.

RAW 264.7 mouse macrophages (ATCC, Manassas, VA) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS).