Linuron

Compound treatment was performed for 24 hours with compounds diluted in DMEM/F12+GlutaMAX that was supplemented with 10% FBS and 1% penicillin/streptomycin. Compounds used in these studies are: 0.2–20 μM linuron (#36141, Sigma-Aldrich, 20 mM stock in EtOH), 20 μM methyl carbamate (#246352, Sigma-Aldrich, 20 mM stock in EtOH), 20 μM naphthalene (#147141, Sigma-Aldrich, 20 mM stock in EtOH), 20 μM vinclozolin (#45705, Sigma-Aldrich, 20 mM stock in EtOH), 20 μM PFNA (#394459, Sigma-Aldrich, 20 mM stock in EtOH), 10 μM ATP (#P0756, NEB, 1:1000), 50 μM BD1063 (#SML0276, Sigma-Aldrich, 50 mM stock in PBS), 100 ng/mL IL-1ß (#401-ML-005, R&D Systems, 100 μg/mL stock in PBS), 50 ng/mL TNFα (#410-MT-010, R&D Systems, 100 μg/mL stock in PBS), 1 μM GSK2656157 (#504651, Calbiochem, 1 mM stock in EtOH), and 50 μM STF083010 (#SML0409, Sigma-Aldrich, 50 mM stock in DMSO).

All mice used were WT animals on the C57Bl/6 background. EAE was induced with 25 μg of MOG_35–55 (#110582, Genemed Synthesis Inc.) mixed with complete Freund’s Adjuvant at a ratio of 1:1 (v/v at a concentration of 5 mg/mL. Mice received 2 subcutaneous injections of 100 μL each of the MOG/CFA mix. Mice then received a single intraperitoneal injection of pertussis toxin (#180, List Biological Laboratories) at a concentration of 2 ng/μL in 200 μL of PBS. Mice received a second injection of pertussis toxin at the same concentration two days after the initial EAE induction. Mice were monitored and scored daily thereafter. EAE clinical scores were defined as follows: 0 – no signs, 1 – fully limp tail, 2 – hindlimb weakness, 3 – hindlimb paralysis, 4 – forelimb paralysis, 5 – moribund, as described previously (Mayo et al., 2014 ; Rothhammer et al., 2016 ). For studies of the effects of Linuron on EAE, mice were injected intraperitoneally with 100 mg/kg of Linuron dissolved in corn oil (#C8267–500ML, Sigma-Aldrich) at a concentration of 12.5 mg/mL on alternating days starting at day 6 post-EAE induction. Sex differences were not analyzed but only a single sex was used within any set of EAE experiments. Mice were randomly assigned to treatment groups.

High-performance liquid chromatography (HPLC)-grade methanol, acetonitrile and pH buffers (4.0, 7.0 and 10.0) were purchased from VWR (Atlanta, GA, USA). Analytical standards for lindane, diazinon, linuron, trifluralin, paraquat and ammonium acetate were purchased form Sigma Aldrich (St. Louis, MO, USA). Sulfuric acid (H₂SO₄, 95–98%), PCP and 2,4,6-TCP were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Ethylene glycol and formic acid (HCOOH, 88%) were purchased from Thermo Fisher (Waltham, MA, USA). CM was obtained from BASF (Lampertheim, Germany) with a total surface area of approximately 850 m²/g, an external surface area of 70 m²/g and cation exchange capacity equal to 97 cmol/kg [28 (link)]. SM clay was obtained from the Source Clay Mineral Repository at the University of Missouri-Columbia with an estimated cation exchange capacity equal to 75 cmol/kg. The generic formula for these clays is: (Na,Ca)_0.3(Al,Mg)₂Si₄O₁₀(OH)_2⋅nH₂O. Clays were sieved through 45 μm to achieve uniform particle size [28 (link),29 (link)]. Ultrapure deionized water (18.2 MΩ) was generated in the lab using an Elga™ automated filtration system (Woodridge, IL, USA) and used in all experiments.