Glutamax 100

The six cell lines, A375, H460, HT29, MCF7, HepG2, and HaCaT (Lonza, Verviers, Belgium), were kept in culture and expanded at 37 °C in a humidified atmosphere of 5% CO₂ in DMEM (Dulbecco’s Modified Eagle’s Medium, Lonza, Basel, Switzerland) culture medium for HaCaT, HepG2, HT29, MCF7, and A375, and RPMI-1640 for H460, supplemented with 10% FBS, Penicillin/Streptomycin 100× (Euroclone, Devon, UK), Glutamax 100× (Invitrogen, Carlsbad, CA, USA) and non-essential amino acids 100× (Invitrogen). Phosphate buffer (PBS, phosphate-buffered saline, Ca²⁺ and Mg²⁺ free) and trypsin (Ca²⁺ and Mg²⁺ free) were supplied by Euroclone. The cells were plated 15 × 10³ perwell in 96-well tissue culture plates and allowed to attach for 24 h. Then, cells were treated with PECS dissolved in culture medium supplemented with 1% FBS at different concentrations for 48 h (50 μg/mL, 80 μg/mL, 110 μg/mL, 140 μg/mL, 170 μg/mL, and 200 μg/mL). PECS were dissolved in sterile H₂O at 100 mg/mL.

Dissociated cortical and hippocampal cultures were prepared as follows: Cortices and hippocampi of (E18) rat embryos were finely chopped. The tissues were digested with 0.125% trypsin (Sigma Aldrich, St. Louis, MO, USA) and 0.05% DNAse (Sigma Aldrich) in Hank’s Balance Salt Solution (Sigma Aldrich) without calcium and magnesium for 18 min at 37 °C. The digestion was halted by adding fetal bovine serum (FBS, 10%, Sigma Aldrich, St. Louis, MO, USA) complemented medium (Neurobasal). This step was followed by mechanical dissociation by trituration. Cells were resuspended in Neurobasal medium (Gibco Invitrogen, Waltham, MA, USA), supplemented with 2% B-27 supplement (Gibco Invitrogen), 1% stable L-Glutamine (GlutaMAX 100× Gibco Invitrogen), 1% PenStrep (Penicillin–Streptomicin Solution, Gibco Invitrogen), and plated on the already poly-L-ornithine- (100 µg/mL, Sigma Aldrich) coated MEAs at the final density of 1′500 cells/mm² and 1′300 cells /mm² for the cortical and hippocampal cultures, respectively. The cultures were maintained at 37 °C with 5% CO₂ and 95% humidity. After 5 days, and twice a week afterward, a half volume of the medium was replaced with BrainPhys medium, supplemented with 2% NeuroCult SM1, 1% Glutamax, and 1% PenStrep solution.

Resolvin D1 (RvD₁, 95%), resolvin D2 (RvD₂, 95%), resolvin D3 (RvD₃, 95%), resolvin D4 (RvD₄, 95%), resolvin D5 (RvD₅, 95%), deuterated resolvin D1 (RvD₁-d₅, 95%), deuterated resolvin D2 (RvD₂-d₅, 95%) and deuterated resolvin D3 (RvD₃-d₅, 95%). Acetonitrile (99.8%) and formic acid (98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-propanol (HPLC grade, 99.9%) from Merck (Darmstadt, Germany). Chloroform (HPLC grade, 99.8%) was obtained from Merck (Darmstadt, Germany). A Millipore Milli-Q system was used to produce ultra-pure water 18 MΩ (Millipore, Milford, CT, USA). Cis-4,7,10,13,16,19-docosahexaenoic acid (DHA, ≥98%) were purchased from Sigma-Aldrich (Oslo, Norway). Leibovitz’s L-15 medium was from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS, cat# 14-801F) was from BioWhittaker (Petit Rechain, Belgium). The glutaMaxTM 100× (Gibco-BRL, cat# 35056) was from Gibco-BRL (Cergy-Pontoise, France).

Delphinidin-3-O-rutinoside standard (D3R, ≥95% purity) was purchased from Extrasynthese (Genay, France). Chlorogenic acid (CA, >98% purity), epicatechin (EC, >90% purity), dimethyl sulfoxide (DMSO), 0.4% trypan blue stain solution, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (FBS), Roswell Park Memorial Institute medium (RPMI-1640), High glucose Dubelcco’s Modified Eagles Medium (DMEM), minimum essential medium non-essential amino acids (MEM NEAA) 100×, GlutaMAX^TM 100×, penicillin/streptomycin 100× solution (10.000 Units mL⁻¹/10.000 µg mL⁻¹), and 0.25% Trypsin-EDTA solution were purchased from Gibco/Life technology corporation (Paisley, UK).

Prostaglandins PGE₂ (99%) and PGE₃ (98%); prostacyclins 6-keto-PGF_1α (98%) and Δ17-6-keto-PGF_1α (98%); deuterated internal standards PGE₂-d₄ (99%) and 6-keto-PGF_1α-d₄ (99%) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Acetonitrile (99.8%), methanol (99.8%) and formic acid (98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A Millipore Milli-Q system was used to produce ultra-pure water 18 MΩ (Millipore, Milford, MA, USA). Cis-5,8,11,14-eicosatetraenoic acid (ARA, 85%) and cis-5,8,11,14,17-eicosapentaenoic acid (EPA, 99%), were purchased from Sigma–Aldrich (Oslo, Norway). Leibovitz`s L-15 medium and laminin (cat#L2020) were from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS, cat# 14-801F) was from BioWhittaker (Petit Rechain, Belgium). The glutaMaxTM 100 × (cat# 35056) and the collagenase type IV (cat#17104019) were from Gibco-BRL (Cergy-Pontoise, France). The penicillin-streptomycin mixture (cat#17-602E) and the trypan blue solution (cat#17-942E) were from Lonza (Falun, Sweden).

Primary neuron cultures were prepared from the cortex of embryonic day 15 (E15) C57BL/6 mice. For the protocol, the brain was harvested, and the meninges and hippocampus were removed from the hemisphere under a microscope. The cortical neurons were collected in modified Eagle’s medium (MEM, Gibco/Thermo Fisher) with 1% glutamate and were washed once. The cortices were then treated with 0.125% trypsin at 37 °C for 10 min. The cell suspensions were filtered through a 40 μm cell strainer and plated on plate wells precoated with poly-D-lysine (P4707; Sigma-Aldrich). The collected cortical cells were counted and seeded at a density of 1.25 × 10⁶ neurons/well for in a 6-well plate and 3 × 10⁵ neurons/well for in a 24-well plate in Neurobasal medium containing 2% B27 supplement, 1% GlutaMAX (100×), and 1% antibiotic–antimycotic solution (all from Gibco/Thermo Fisher). One day after seeding the cells, the medium was replaced, and culture medium was then partially changed once every 3–4 days with freshly supplemented Neurobasal medium. The cultures were maintained at 37 °C in a humidified incubator of atmosphere mix containing 5% CO₂.

C. pecorum 1710S (isolate from a swine abortion) was kindly provided by Professor Storz, Baton Rouge, LA, USA [3 (link)] and propagated in HeLa cells. Crude C. pecorum stock was generated by mechanical disruption (scraping) of infected HeLa cells into infection medium, sonication (Branson Sonifier 250; Branson Ultrasonics, Danbury, CT, USA) on ice, centrifugation of infectious particles from the medium at 10,000 g at 4°C for 45 minutes, and suspension in SPG medium. Stock was stored at −80°C and frozen stock aliquots were thawed immediately before infections were carried out. SPG medium consisted of 218 mM sucrose (Sigma-Aldrich), 3.76 mM KH₂PO₄ (Sigma-Aldrich), 7.1 mM K₂HPO₄ (Merck Eurolab AG, Dietlikon, Switzerland), and 5 mM GlutaMAX-100 (GIBCO).