Cacl2

ATP, ADP, AMP, adenosine, dipyridamole, apyrase and dimethylsulfoxid (DMSO) were purchased from Sigma (Taufkirchen, Germany); 2-methylthioadenosine diphosphate trisodium salt (2meSADP) was from Tocris (Bristol, UK); DMEM, Dulbecco’s Phosphate-Buffered saline without Ca²⁺ and Mg²⁺, trypsin/EDTA solution, HEPES was obtained from Thermo Scientific (Walldorf, Germany); NaCl, KCl, MgCl₂, CaCl₂, K₂HPO₄, NaHCO₃, D-glucose, paraformaldehyde (PFA), Triton X-100, Tween 20, bovine serum albumin (BSA), and sucrose were from Roth (Karlsruhe, Germany). Heparin (Heparin-Natrium-250 000-ratiopharm) was from Ratiopharm (Ulm, Germany), Narcorene^® from Merial GmbH (Hallbergmoos, Germany), ticagrelor was purchased from Cayman Chemical (Hamburg, Germany); concentrated stock solutions of ticagrelor and dipyridamole were prepared in DMSO and diluted in artificial cerebrospinal fluid (ACSF) or cell culture medium right before use.

The polyether sulfone (PES) ultrafiltration membrane modules, comprising 10 Multibore^® fibers, each containing 7 capillaries with an inner diameter of 0.9 mm, were supplied by inge GmbH (DuPont, Greifenberg, Germany). Each membrane module has an overall membrane surface of 0.05 m² and molecular weight cut-off (MWCO) of 100 kDa. Polydiallyldimethylammonium chloride (PDADMAC) (20% solution with a molecular weight of 250–350 kDa) and poly-(4-styrenesulfonic acid) (PSS) (molecular weight of 1000 kDa) were purchased from Sigma Aldrich (Schnelldorf, Germany). NaCl, MgSO₄, CaCl₂ and NaOCl were purchased from Carl Roth GmbH (Karlsruhe, Germany). All salts were of analytical grade.

Tenfold dilution series of SARS-CoV-2 containing solutions in PBS supplemented with 1% (v/v) P/S, 0.6% (v/v) BSA (35%) (Sigma-Aldrich), 0.01% (w/v) CaCl₂ (Roth), and 0.01% (w/v) MgCl₂ (Roth) was prepared to infect VeroE6 cells grown to a confluent monolayer in 6-well plates. 1 h.p.i. at 37 °C the virus inoculum was replaced by plaque medium composed of 63% (v/v) 2 × MEM ((20% (v/v) 10 × MEM (Gibco), 3.2% (v/v) NaHCO₃ (7.5%) (Gibco), 2% (v/v) HEPES (1 M; pH 7.2) (Sigma-Aldrich), 1.2% (v/v) BSA (35%), 1% (v/v) 100 × Penicillin/Streptomycin/l-Glutamine solution (10,000 U/ml Penicillin; 10,000 µg/ml Streptomycin; 29.2 mg/ml l-Glutamine) (Gibco)), 2% (v/v) FBS, and 35% (v/v) Agar (2%) (Oxoid). Plaques were counted after 72 h incubation at 37 °C.

Prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂), leukotriene B₄ (LTB₄), 6-trans-leukotriene B₄ (t-LTB₄), 6-trans-12-epi-leukotriene B₄ (trans-epi-LTB₄), 12-epi-leukotriene B₄ (epi-LTB₄), 20-hydroxyleukotriene B₄ (20-OH-LTB₄), thromboxane B₂ (TxB₂), 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE), prostaglandin B₁ (PGB₁), prostaglandin F_2α (PGF_2α), and zileuton were purchased from Cayman Chemicals (Ann Abor, USA). MS-grade methanol (MeOH), isopropanol (IPA), and acetonitrile (ACN) were from VWR (Darmstadt, Germany), ammonium hydroxide from Honeywell (Charlotte, USA), and formic acid, diclofenac, and acetylsalicylic acid (ASS) from Merck KGaA (Darmstadt, Germany). Lipopolysaccharide (LPS), Histopaque-1077^®, n-hexane, and methyl formate were from Merck (Darmstadt, Germany). L-glutamine and CaCl₂ were purchased from Carl Roth (Karlsruhe, Germany).

Briefly, 14.6 g calcium chloride (CaCl₂, Carl Roth, Karlsruhe, Germany), 14.3 g disodium hydrogenphosphate (Na₂HPO₄, Carl Roth, Germany), and 0.8 g sodium fluoride (NaF, Carl Roth, Germany) were mixed in dry state, and 40 mL of MilliQ water were added shortly before ultrasonication [43 ] for 5 min at an energy intake of 18 kJ using a Sonoplus Ultrasonic Homogenizer (Bandelin, Berlin, Germany) and a KE76 probe. Particles were washed with MilliQ water and air dried at 50 °C overnight.
FAp particles were coated in an aqueous solution using eADF4(κ16) dissolved in 6 M guanidinium thiocyanate and dialyzed against 10 mM Tris buffer, pH 7.5 for 16 h. The coating was performed using 10 mg particles in 1 mg/mL protein solution for 8 h, followed by centrifugation at 13,000 rpm for 10 min and washing in MilliQ water. κFAp refers to FAp particles coated with eADF4(κ16).

OSCs were prepared as described by [45 (link)]. In brief, brains from 2 to 3 month old C57BL/6 mice were removed and immediately immersed in ice-cold Ringer solution [2.5 mM KCl (Merck KGaA, Darmstadt, Germany), 1 mM MgCl₂ (Sigma-Aldrich), 260 mM d-Glucose (Applichem), 26 mM NaHCO₃ (Merck KGaA), 1.25 mM NaH₂PO₄ (Merck KGaA), 2 mM pyruvic acid (PAA Laboratories), 3 mM myo-inositol (Sigma-Aldrich), 1 mM kynuric acid (Sigma-Aldrich), 2 mM CaCl₂ (Carl Roth GmbH), pH 7.3]. The brains were embedded in 4% low melting agarose (Sigma-Aldrich). After polymerization agarose blocks were trimmed and glued onto the cutting table of a vibratome (VT1000, Leica, Heerbrugg, Switzerland). Brains were cut in coronal slices of 250 µm. Slices were then collected and stored in ice-cold Ringer solution before floating onto semi-porous membrane inserts (Millipore, 0.4 mm pore diameter) according to Stoppini et al. [46 (link)]. The slices were cultivated in wells of a 6-well plate at 37°C under 5% CO₂ in a standard medium consisting of DMEM/Ham’s F12 (pH 7.3; PAA Laboratories), 24% normal horse serum (PAA Laboratories), 2% HEPES (PAA Laboratories), 0.1% gentamycin (PAA Laboratories) and additional 10 mM d-glucose (Sigma-Aldrich). Medium was changed every other day and viability of brain slices was assessed using PI staining.

DON was obtained from Romer Labs (Tulln, Austria) and CER was purified as previously described [28 (link)]. Cell culture media and supplements were obtained from Gibco® Life Technologies (Karlsruhe, Germany). KH₂PO₄, KCl, NaCl, Na₂HPO₄, D-glucose, HEPES, CaCl₂, MgCl₂, glycin, formaldehyde, sodium dodecyl sulfate (SDS), NaOH and Triton X-100 were purchased from Carl Roth (Karlsruhe, Germany) and Lucifer Yellow CH di-lithium salt from Santa Cruz Technologies (Dallas, TX, USA). For LC-MS/MS measurements, MS grade water was purchased from VWR (Fontenay-sous-Bois, France), acetonitrile (ACN) and methanol (MeOH) from Honeywell (Seelze, Germany). Acetic acid was obtained from Merck (Darmstadt, Germany) and ammonium acetate (both LC-MS grade) from Sigma Aldrich (Vienna, Austria). DON-3-sulfate, DON-15-sulfate and DON-3-glucuronide were kindly provided by Dr. Philipp Fruhmann (Technical University of Vienna) and synthesized as previously described [44 (link)].