Resveratrol

The conjugation of highly purified human fibrinogen with fluorescein isothiocyanate (FITC) via FITC-celite was performed as previously described^{64 (link)}. Sytox-Green was from Molecular Probes and Hoechst33342 from Life Technologies. Anti-CD42a-PE, mouse IgG clone Beb1, anti-CD62P-FITC, PAC-1-FITC, and CD16b were from BD Pharmingen. Anti Mac-1 activation-dependent epitope antibody (CBRM1/5) was from eBioscience. Anti-human defensin antibody was purchased from Hycult Biotech and anti-FDP-lysine antibody was from Abcam. Anti-mouse IgG-FITC (Sigma-Aldrich) was used as secondary antibody. Resveratrol (≥98% trans-3,4′,5-Trihydroxystilben) was purchased from Carl Roth, human antithrombin III from Enzyme Research Laboratories, human serum, taurine, phorbol 12-myristate 13-acetate (PMA), bovine thrombin, ADP, Thrombin Receptor Activator Peptide 6 (TRAP-6) and α1-antitrypsin were from Sigma-Aldrich and reduced glutathione for i.v. injection (Tationil®) was from Roche (Italy). IgG depleted human serum was from Sunnylab. RPMI 1640 medium was from Biochrom. Melagatran (for injection) was from Astrazeneca (Wedel, Germany). Recombinant staphylokinase (SAK) was kindly provided by Dr. Bernhard Schlott (Leibnitz Institute for Age Research, Jena, Germany) and was prepared as previously described^{65 (link)}.

N-hippuryl-His-Leu (Hip-His-Leu) (PubChem CID: 94418), ACE (peptidyl-dipeptidase A, E.C. 3.4.15.1) (PubChem CID: 329770629), glutathione-S transferase from horse liver (PubChem CID: 114886), monochlorobimane (PubChem CID: 114886), 2′,7′-dichlorofluorescein diacetate (PubChem CID: 24894058), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH, PubChem CID: 57654141), N-1-naftiletilendiamine (PubChem CID: 329754555), HPLC grade acetonitrile and trifluoroacetic acid were provided by Sigma Aldrich (Madrid, Spain). Heparin (PubChem CID: 772) was provided by DeltaLab (Barcelona, Spain). Quercetin, gallic acid, (+)-catechin, p-coumaric acid and (−)-epicatechin were purchased from Fluka/Sigma-Aldrich; caffeic acid, malvidin-3-O-glucoside, vanillic acid, procyanidin dimer B2 and 4-hydroxybenzoic acid were purchased from Extrasynthése (Lyon, France); resveratrol was purchased from Carl Roth (Karlsruhe, Germany); cyanidin-3-O-rutinoside, ferulic acid and peonidin-3-O-rutinoside were purchased from PhytoLab (Vestenbergsgreuth, Germany). The rest of chemical solvents used in this study were of analytical grade.

The human microglia (HMC3) and astrocytes (SVGA) were seeded two days prior to stimulation to adhere at 5% CO₂/37 °C. On the day of the experiment, the cells were washed once with phosphate-buffered saline (PBS). The medium was renewed for the controls, and for the treatment group, changed to 1 mL of OGD medium, a DMEM-based glucose-free medium (Cat. A14430, DMEM no-glucose, Thermo Fisher Scientific), with 1% penicillin–streptomycin (10,000 U/mL; Thermo Fisher Scientific) and 3.97 mM L-glutamine (Carl Roth), supplemented or unsupplemented with 100 µM of resveratrol (Cat. R5010, Sigma-Aldrich/Merck, Taufkirchen, Germany) dissolved in polyethylene glycol 400 (PEG400, Caesar & Loretz GmbH, Hilden, Germany). The PEG400 did not have a significant effect on the cells used in this study (Table S1, Supporting Information). The cells were kept at 3% O₂/5% CO₂/37 °C for the times indicated for each experiment in an incubator (Memmert ICO150 with AtmoCONTROL, Schwabach, Germany). Afterward, the media and cells were collected for subsequent analysis.