Rpmi 1640

Anti-ERK1/2 (Ab-202/204) and Anti-ERK1/2 (Phospho-Thr202/Tyr204) and
anti-β-actin were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA.
Clusterin (A-9) (sc-166907, 1:200) was purchased from Santa Cruz (Shanghai,
China). Horseradish peroxidase-conjugated goat anti-mouse and donkey anti-rabbit
antibodies were obtained from Amersham Biosciences. DMEM/F12, RPMI 1640, FBS, and
Normocin antibiotic were all purchased from InvivoGen. PD98059, a specific
inhibitor of ERK kinase was purchased from Biovision, Beijing, China. Mitogen
activated kinase 1 human recombinant (MEK1), the ERK activator kinase 1/2(ERK1/2),
was from Shanghai, China. Cisplatin (DDP) was purchased from Zibo.co, Shandong,
China.

Production of ROS was determined by a real-time luminol-based luminescence assay, as previously described [32 ], with minor modifications. Briefly, HKLs were seeded in RPMI-1640 (Gibco) supplemented with 100 IU p/s at a density of 1 × 10⁶ per well and incubated for 60 min at 27 °C (Nile tilapia) or at 19 °C (rainbow trout) in white 96-well plates (CLS3912; Corning). Subsequently, cells were stimulated with one of the following: RPMI-1640 cell culture medium (negative control), zymosan (positive control; tlrl-zyd, 50 µg mL⁻¹, InvivoGen) or one of the MSP-rich extracts (Table 1) at concentrations of 250, 500, 750, 1000 and 1500 µg mL⁻¹. Per independent experiment, all stimuli were performed on HKLs isolated from the same animal. For each independent experiment, each condition was tested in triplicate. For ROS analysis, n = 4 independent experiments (n = 4 fish) per species was performed. Chemiluminescence emission was measured in real time (every 2 min for 120 min) with a FilterMax F5 Multi-Mode Microplate Reader at 27°C (Nile tilapia) or at 19 °C (rainbow trout), and expressed as fold changes based on areas under the curve [33 (link)]. Fold changes were based on data from stimulated relative to unstimulated HKLs (treated with RPMI).

pLAg was prepared by suspending promastigotes of the L. panamensis strain MHOM/COL/81/L13 at a concentration of 1 × 10⁷ parasites/mL, freezing in liquid nitrogen and thawing at 37°C four times. Cells were suspended in RMPI 1640 (Sigma-Aldrich) with 10% FBS (Gibco, Carlsbad, CA), 2 mM L-glutamine, penicillin (100 U/mL) and streptomycin (100 mg/mL) and distributed in 96 well plates in 200 μL per well. For PBMC cultures, 4 × 10⁵ PBMCs and for B cell/CD4 T cell co-cultures, 2 × 10⁵ B cells or 2 × 10⁵ T cells or both cell types were plated per well. Then 8 μL of pLAg were added to the appropriate wells to reach a parasite:cell ratio of 0.2:1. The cells were incubated for 5 days at 37°C with 5% CO₂. Cells and supernatants were harvested for evaluation of cell surface markers and cytokine secretion, respectively.
Ramos cells were cultured in RPMI 1640 with 10% heat-inactivated FBS at a concentration of 10⁶ cells/mL with pLAg (10⁷ parasites/mL final concentration), 5 μM CpG ODN 2006 (InvivoGen, San Diego, CA) or no stimulus for 48 hours. Cells were harvested for evaluation of cell surface markers or BCR-mediated endocytosis.

Glycolytic and mitochondrial activity of monocytes was measured using a Seahorse extracellular flux 96 analyzer (Seahorse Bioscience). Monocytes were seeded at a density of 4 × 10⁵ cells/100 µl RPMI1640 (#61870, Gibco, Thermo Fisher) without FCS and adhered for 30 min at cell culture conditions. After two washing steps, 180 µl RPMI 1640 (R1383, Sigma-Aldrich) containing 5% FCS, 2 mM glutamine, and 5 mM glucose or without glucose was added. The injection ports were filled with RPMI 1640 without FCS plus the indicated stimuli or inhibitor (LPS-EB ultrapure from E. coli 0111:B4 strain 100 ng/ml, Invivogen; apocynin 1 mM, Sigma-Aldrich; 2-deoxyglucose (2-DG) 5 mM, Sigma-Aldrich; etomoxir 250 µM, Sigma-Aldrich; BPTES 30 µM, Sigma-Aldrich; dorsomorphin/compound C 10 µM, Abcam). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined at 37°C. The parameters of oxidative phosphorylation were analyzed following the injection of the inhibitors oligomycin (1 µM; Cayman Chemicals), FCCP (2 µM; Cayman Chemicals), Rotenone (1 µM; Sigma-Aldrich), and antimycin A (1 µM; Sigma-Aldrich).

Adherent spleen cells or peritoneal macrophages from C57BL/6 (WT) or TLR2 knockout (KO) mice were used to assess IL-12p40 and IL-10 production in response to stimulation with ArtinM. Spleen cells (1×10⁷/mL) were distributed in a 24-well microplate and cultured at 37°Cfor 2 h in a humidified atmosphere with 5% CO₂. Cells were washed with RPMI 1640 and incubated with ArtinM (156 nM) or Pam3CSK4 (P3C4, 1 µg/mL) (Invivogen) for 24 h. Peritoneal macrophages (2×10⁶/mL) from WT and TLR2-KO mice were also assayed. Cells were plated in a 96-well microplate and cultivated for 12 h under conditions similar to those adopted for adherent spleen cells. Peritoneal macrophages were incubated for 48 h with ArtinM (39 nM) or P3C4 (1 µg/mL). The amount of IL-12 p40 and IL-10 in the culture supernatants was measured by ELISA, using specific antibody pairs purchased from Pharmingen (San Diego, CA) in accordance with the manufacture's protocol. Murine recombinant cytokines were used to create standard curves. IL-12 p40 and IL-10 cytokine concentrations were determined with reference to the standard curves.

Eosinophils and neutrophils were collected from whole blood using MACSxpress Neutrophil Isolation Kit, human and Eosinophil Isolation Kit, human, (Miltenyi Biotec) according to the manufacturer’s instructions. Remaining erythrocytes were lysed. Cells were diluted in Complete Medium (CM) consisting of RPMI 1640 (Invivogen, San Diego, CA, USA) with 10% autologous plasma, penicillin 100 U/mL and streptomycin 100 μg/mL (Invitrogen, Carlsband, CA, USA), to a concentration of 1 × 10⁵cells/ml. Migration set up was conducted with transwell plates (3.0 μm PTFE Collagen Coated Membrane, Corning, NY, USA). Cells were added (5 × 10⁵ neutrophils in the bottom well and 2.5 × 10⁵ eosinophils in the insert) and incubated for 3 h at 37 °C in a humidified 5% CO₂ air atmosphere. Inserts were removed and cell suspension in the wells were analysed with flow cytometry using CountBright TM Absolute Counting Beads (Life Technologies, Eugene, OR, USA).