Activated charcoal

Hydroxyproline content of mouse liver tissue was measured as a marker of net ECM deposition as previously described [38] (link). Briefly, duplicate samples of liver tissue (60 mg) were hydrolysed in 1.5 ml of 6 M HCl at 110°C overnight. Cooled samples were diluted to 6 ml in dH₂O and adjusted to pH 7.4 before incubation with activated charcoal (Ajax Finechem). After 30 mins the samples were filtered (Whatman No. 3) and further diluted to 12 ml in dH₂O. Two hundred μl sample were combined with 400 µl isopropanol and 200 µl chloramide T (308 mM) for 5 mins. Ehrlich's solution (2.5 ml) was added and the samples were incubated at 65°C for 25 mins before being cooled. Sample aliquots of 200 µl were then transferred to a 96-well plate and the absorbance was measured at 570 nm (POLARstar Omega; BMG Labtech). Hydroxyproline concentration was calculated using a hydroxyproline standard (Fluka Chemicals) and normalised for starting tissue weight.

Δ⁹-THCA was isolated from hemp extracts. In brief, crude cannabis extract was dissolved in methanol (LiChrosolv^®; Merck, Darmstadt, Germany) and treated overnight with activated charcoal (Ajax Finechem, Wollongong, Australia) at 4°C. The solution was filtered through a Büchner funnel and the filtrate was collected. The solvent was removed under pressure, and then reverse phase column chromatography (Büchi Reveleris PREP; Büchi AG, Flawil, Switzerland) with a C18 column (Büchi AG) was used to purify the residue and elute Δ⁹-THCA. Purity of Δ⁹-THCA isolated was 97% with 3% Δ⁹-THC. In addition, we purchased Δ⁹-THCA-A with a purity of 99.5% (<0.5% Δ⁹-THC content) and Δ⁹-THC (dronabinol, 100% purity) from THC Pharm GmbH (Frankfurt, Germany). Cannabinoids were stored protected from light at −30°C. Sodium valproate was purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Analytical standards were purchased from Novachem Pty Ltd (Heidelberg West, Australia).

PLDP was obtained from YAEGAKI Bio-industry, Inc. (Himeji, Japan). The extracted PLDP components have been previously reported⁵ . Charcoal-Dextran stripping of PLDP prepared as follows. Activated charcoal (Fisher Chemicals, D127–500, 10 g/L) and Dextran (Sigma, D8821, 1 g/L) was added to PLDP (15 mL) followed by mixing at 56 °C for 45 min and centrifugation at 4000 rpm for 20 min and the supernatants were test for cell morphological change. A PL kit including LPC, LPE, phosphatidylinositol (PI), sphingomyelin (SM), PS, LPS, PC, PE, cardiolipin (CL), phosphatidic acid (PA), LPS, and CB was purchased from Olbracht Serdary Research Laboratories (Toronto, Canada). Murine microglial BV-2 and SIM-A9 cells were provided from Dr. Hiroshi Ueda (Kyoto University). Fetal bovine serum, poly-D-lysine, penicillin-streptomycin, and other chemicals were purchased from Sigma (St. Louis, MO, USA). Anti-ATX (GTX106209) antibody was purchased from GeneTex Inc. (Irvine, CA, USA). Anti-β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 18:1 lyso NBD-PC was purchased from Avanti Polar Lipids (Alabaster, AL, USA). The ATX inhibitor BI-2545 was provided by Boehringer Ingelheim (opnMe).

Glucose (C₆H₁₂O₆), conc. sulfuric acid (98% H₂SO₄), potassium hexacyanoferrate(III) (C₆FeK₃N₆), and potassium chloride (KCl) were obtained from Fisher chemicals. Malathion (C₁₀H₁₉O₆PS₂) was obtained from Insecticides India Limited. Disodium phosphate (Na₂HPO₄·H₂O), monosodium phosphate (NaH₂PO₄), sodium hydroxide (NaOH), ethanol (C₂H₅OH), and isopropyl alcohol (C₃H₈O) were procured from Rankem chemicals. Nafion (C₉HF₁₇O₅S) and activated charcoal were taken from Fisher Scientific. For all experimental work and the preparation of stock solutions, deionized (DI) water was used.