Penicillin

RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC TIB-71, Manassas, VA; Lots 70000171, 70012232 with cell identity confirmed by ATCC via growth properties, morphology, and COI assay). Cell media (supplemented DMEM) was composed of DMEM obtained from Thermo Fischer Scientific (Waltham, MA), fetal bovine serum (FBS, contains ~400 μM BSA as specified by manufacturer) from Millipore-Sigma (St. Louis, MO), penicillin, streptomycin and GlutaminePlus (L-alanyl-L-glutamine) from Atlanta Biologicals (Flowery Branch, GA). Dulbelcco's phosphate buffered saline (D-PBS) was from Gibco (Gaithersburg, MD). All cell media was regularly subjected to mycoplasma testing to ensure lack of infection. Matriplate 96 well imaging plates were from Matrical, Inc. (Spokane, WA). Acetylsalicylic acid (Lot SLBV2290), ibuprofen (Lot SLBX6584), acetaminophen (Lot SLBR2060V), diclofenac (Lot BCBS5961V), wortmannin (Lot 086M4026V), PDGF (Lot SLB63548V), and HEPES (free acid) were obtained from Millipore-Sigma (St. Louis, MO). Gö6976 (Lot 6A/174381) was purchased from Tocris Biosciences (Minneapolis, MN). CellMask Deep Red and Green membrane stains were purchased from ThermoFischer Scientific (Waltham, MA). Cell-culture grade DMSO was purchased from MP Biomedicals (Santa Ana, CA). AKT-PH-mRFP mammalian expression plasmid was subcloned by John H. Evans [26 (link), 30 (link)].

Primary ependymal cell cultures were established as described previously (Delgehyr et al., 2015 (link); Guirao et al., 2010 (link)). Briefly, brains of postnatal day 0–3 transgenic Arl13B-EGFP^tgmice (Delling et al., 2013 (link)) or wild type mice were collected, telencephala dissected, the choroid plexus and meninges removed, the tissue enzymatically digested, cells mechanically dissociated, plated and grown to confluence. Confluent cell layers were trypsinized, and replated on poly-L-lysine coated coverslips at a density of 10⁷ cells/ml in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, GlutaMAX supplement, and pyruvate (Life Technologies), 10% decomplemented fetal bovine serum (FBS, Atlanta Biologicals), and 100 units/ml penicillin/100 μg/ml streptomycin (Atlanta Biologicals), which was replaced by serum-free media the next day. Ependymal cells were cultured for up to two weeks.

Chinese hamster ovary (CHO-K1) cells and a murine pituitary cell line (GH3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in culture at 37 °C, 5% CO₂ per the supplier’s recommendations. CHO-K1 cells were cultured in Ham’s F12K medium supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Norcross, GA), 100 IU/mL penicillin, and 0.1 μg/mL streptomycin. GH3 cells were cultured in Ham’s F12K medium supplemented with 2.5% FBS, 15% horse serum (ATCC), 100 IU/mL penicillin, and 0.1 μg/mL streptomycin. The media and its components were purchased from Mediatech Cellgro (Herndon, VA). In the passage immediately preceding experiments, cells were transferred onto glass coverslips pretreated with poly-L-lysine to improve cell adhesion.
The composition of solutions utilized in different experiments is described in respective sections below. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and Life Technologies (Grand Island, NY). The osmolality of the solutions was between 290 and 310 mOsm/kg, as measured by a freezing point microosmometer (Advanced Instruments, Inc., Norwood, MA). All experiments were performed at room temperature (22 ± 2 °C).

Trypsin, penicillin, streptomycin, heat-inactivated fetal bovine serum, horse serum and soybean Trypsin inhibitor were obtained from Atlanta Biologicals (Norcross, GA, USA). Minimum essential medium, deoxyribonuclease (DNase), poly-l-lysine, cytosine arabinoside, Hoechest-33342, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX), 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KBR-7943), nifedipine, fluorescein diacetate, propidium iodide, dichloromethane and polyvinylpyrrolidone were from Sigma (St. Louis, MO, USA). Tetrodotoxin (TTX) was purchased from Tocris Cookson, Inc. (Ellisville, MO, USA). Anti-stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) antibody and anti-phospho-SAPK/JNK (Thr183/Tyr185) were from Cell Signaling Technology (Danvers, MA, USA). Anthra[1-9-cd]pyrazol-6(2H)-one (SP 600125), 4-(4-Fluorophenyl)-2-[4-(methylsulfinyl) phenyl]-5-(4-pyridyl) 1H-imidazole (SB 203580), 1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene (U0126) and N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) were from Biomol International (Plymouth Meeting, PA, USA).

Balb/c mice weighing 20–25 g were infected with 130 cercariae of S. mansoni released from experimentally infected B. pfeifferi by the tail and leg immersion technique. After 49 days, adult worms were recovered from mice's mesenteric veins and liver by perfusion with a sterile saline solution (0.9% NaCl) [38 (link)]. Freshly harvested worms were washed three times in a Glasgow Minimum Essential Medium (GMEM) (Sigma, St Louis, USA) supplemented with an antibiotic-antimycotic solution (10,000 U/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B) (Atlanta Biologicals, Lawrenceville, USA) and gentamicin (40 μg/mL).
The bioassay followed the standard operating procedures that recommended at least five female and five male worms per well [39 (link)]. Therefore, 10 adult worms of both sexes were transferred to each well of a 24-well culture plate with 1900 μL of complete GMEM, pH 7.5 (GMEM with 20 mM of HEPES, 40 μg/mL gentamicin, 50 μg/mL penicillin, 50 μg/mL streptomycin, 100 μg/mL neomycin, 2 mM of L-glutamine, and 5% heat-inactivated fetal bovine serum). The plates were then incubated for 2 hours at 37°C in a humid atmosphere of 5% CO₂ to allow acclimatization.