BMDCs were prepared from bone marrow cells by culturing with 20ng/ml GM-CSF (Peprotech) for 7 days. OVA-specific naïve CD4+ T cells were purified from splenocytes of OT-II mice with EasySep™ Mouse Naïve CD4+ T Cell Isolation Kit (STEMCELL). T cells were stained with CellTrace™ Far Red cell proliferation dye (Invitrogen) following manufacturer’s instructions. 2.5×104 BMDCs and 5×104 purified CD4+ T cells were mixed in full RPMI 1640 medium (200μl/well) with 100nM OVA 323–339 peptide (ISQAVHAAHAEINEAGR) or indicated amount of zymosan-conjugated OVA protein and 1mM aminoguanidine hemisulfate. After 3 days co-culturing, supernatants were assayed for IL-17A, IFNγ, IL-22 and IL-2. Cells were harvested for proliferation (T cells) or surface expression of CD86 and pMHC-II (BMDCs) analysis by flow cytometry. To prepare zymosan-conjugated OVA, 20mg/ml zymosan (InvivoGen) was incubated in 8% glutaraldehyde (Sigma) for 1h, then conjugated with 1mg/ml ovalbumin for 1h at 21°C.
Zymosan
Manufactured by InvivoGen
Sourced in United States, France, Germany, Norway
Zymosan is a cell wall preparation derived from Saccharomyces cerevisiae (baker's yeast). It is composed of a complex mixture of polysaccharides, including β-glucans and mannans. Zymosan is commonly used as a tool for stimulating innate immune responses in vitro and in vivo.
Automatically generated - may contain errors
Lab products found in correlation
Pam3csk4, by InvivoGen (16 mentions)
Poly 1 c, by InvivoGen (7 mentions)
Lipopolysaccharides (lps), by Merck Group (6 mentions)
Fsl 1, by InvivoGen (5 mentions)
Flagellin, by InvivoGen (5 mentions)
Imiquimod, by InvivoGen (5 mentions)
Curdlan, by InvivoGen (5 mentions)
Lipopolysaccharides (lps), by InvivoGen (5 mentions)
Pam2csk4, by InvivoGen (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharide lps, by InvivoGen (3 mentions)
Sp600125, by Merck Group (3 mentions)
Depleted zymosan, by InvivoGen (3 mentions)
Luminoskan ascent, by Thermo Fisher Scientific (3 mentions)
Resiquimod, by InvivoGen (2 mentions)
Cpg a, by InvivoGen (2 mentions)
Lipoteichoic acid lta, by InvivoGen (2 mentions)
Nigericin, by Merck Group (2 mentions)
Laminarin, by InvivoGen (2 mentions)
68 protocols using zymosan
1
Antigen-specific T cell activation by BMDCs
2
Monocyte Response to Immune Stimuli
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Monocytes from NFAM1+/+ and NFAM1-/- mice (isolated as described above) were resuspended in cell culture media comprised of RPMI, 10% fetal bovine serum and 1% Penicillin-Streptomycin (all from Life Technologies). Monocytes were cultured in a 96 well tissue culture plate (Corning) at a concentration of 20,000 cells per well and either left untreated or pretreated for one hour with 100 µg/mL of mouse IFN-γ (R&D Systems) and subsequently stimulated with LPS (Sigma), MegaCD40L (Enzo), HKEB, HKLM, HKST, Pam3CSK4, Zymosan, FSL-1, or MDP (all from Invivogen). 24-48 hours post stimulation, supernatant was collected, and cells were harvested by flushing wells with cold PBS. Cells were analyzed by flow cytometry (see details above) and supernatants were analyzed by MSD for the presence of TNF-α (catalog #K152BHB-4), IL-6, IL-12, MIP-1α and MIP-1β (MSD custom U-plex).
3
Zymosan-Induced Peritoneal Inflammation
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Zymosan (0.1 mg/mouse; InvivoGen, Toulouse, France) was injected into the peritoneum of mice for all tested groups, except for the baseline group (time point 0 h). Tr14 at 3 mL/kg (high dose), diluted with 0.9% NaCl to a concentration of 1.5 mL/kg (low dose), and 0.9% NaCl as vehicle were administered i.p. once daily for 6 days before Zymosan injection (pre-treatment) for experimental setting (1) and i.p. 4 h and again 8 h after Zymosan injection (post-induction) for experimental setting (2). At 4, 8, 24, 192 or 360 h after Zymosan injection, mice were euthanized. The inflammatory exudate was obtained by washing the peritoneal cavity with 2 mL PBS. Cells in the exudates were immediately counted using Scepter 2.0 cell counter (Merck Millipore, Burlington, MA, USA) and labeled for subpopulation identification and quantification by Macs quant analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany).
4
PAMP-induced cytokine production in HEC-1A cells
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HEC-1A cells were stimulated with different PAMPs (pam 3CSK4, lipoteichoic acid, poly(I:C), Escherichia coli lipopolysaccharide, FSL-1, flagellin, imiquimod, zymosan) (InvivoGen, San Diego, CA, USA) diluted in cell culture media with incubation at 37 °C in 5% CO2 for 24 h. PAMPs were titrated to determine the optimal concentration for cytokine stimulation. Blank samples contained media only. After 24 h, the supernatant was collected for cytokine detection. Cytokine levels were determined using a Magnetic Luminex® Performance Assay kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Analyte-specific antibodies were pre-coated onto colour-coded microparticles and detection was carried out with a biotinylated antibody cocktail specific to the target cytokines and a streptavidin-phycoerythrin conjugate. Samples were resuspended in 100 µL of Washing Buffer and measured for a phycoerythrin-derived signal using a Luminex® 100 instrument.
5
Modulation of Placental Inflammation by OleaVita
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Experiment 1: Human placental tissues were incubated with control or OleaVita (0.1 or 1 mg/mL) for 6 h at 37 °C. In preliminary experiment, we checked the appreciate dose of OleaVita (0.01, 0.1, 1, or 2 mg/mL) and incubation time (6 or 24 h). Supernatant and tissues were collected for enzyme-linked immunosorbent assay (ELISA), western blotting, and real-time Reverse transcription polymerase chain reaction (RT-PCR), and were stored at −20 °C or −80 °C before use.
Experiment 2: To investigate the role of Jun N-terminal kinase (JNK), human placental tissues were incubated with OleaVita (1 mg/mL) with or without SP600125 (a JNK inhibitor, 10 μM, Merck Millipore, Burlington, MA, USA), for 6 h at 37 °C. Supernatant and tissues were collected for ELISA and were stored at −20 °C before use.
Experiment 3: To investigate the role of OleaVita on toll-like receptor (TLR) ligand-induced inflammatory responses, human placental tissues were pre-incubated with or without OleaVita (1 mg/mL) for 1 h. Tissues were then incubated with zymosan (TLR2 agonist, 50 μM, Invivogen, Carlsbad, CA, USA), lipopolysaccharide (LPS, TLR4 agonist, 1 μg/mL, Sigma-Aldrich), or imiquimod (TLR7 agonist, 20 μM, Invivogen) for 6 h at 37 °C. Supernatant and tissues were collected for ELISA and were stored at −20 °C before use.
Experiment 2: To investigate the role of Jun N-terminal kinase (JNK), human placental tissues were incubated with OleaVita (1 mg/mL) with or without SP600125 (a JNK inhibitor, 10 μM, Merck Millipore, Burlington, MA, USA), for 6 h at 37 °C. Supernatant and tissues were collected for ELISA and were stored at −20 °C before use.
Experiment 3: To investigate the role of OleaVita on toll-like receptor (TLR) ligand-induced inflammatory responses, human placental tissues were pre-incubated with or without OleaVita (1 mg/mL) for 1 h. Tissues were then incubated with zymosan (TLR2 agonist, 50 μM, Invivogen, Carlsbad, CA, USA), lipopolysaccharide (LPS, TLR4 agonist, 1 μg/mL, Sigma-Aldrich), or imiquimod (TLR7 agonist, 20 μM, Invivogen) for 6 h at 37 °C. Supernatant and tissues were collected for ELISA and were stored at −20 °C before use.
6
Murine Bone Marrow-Derived Dendritic Cell Generation
7
PBMC Isolation and Stimulation Protocol
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Blood samples were collected in tubes with lithium heparin (Vacutainer®) and diluted with an equal volume of warm PBS (Gibco, Invitrogen, Massachusetts). PBMCs were isolated by centrifuging at 800 g for 20 min at 18–20℃ on Biocoll separating solution (Biochrom AG, Germany). PBMCs were washed three times, and the cell pellet was resuspended in complete medium [RPMI 1640 with HEPES 25 mM and l ‐Glutamine (Gibco, Life Technologies Ltd, UK), supplemented with 10 ml/L Penicillin‐Streptomycin USA, 50 μl/L 1 M β‐mercaptoethanol, 20 ml/L l ‐Glutamine plus MEM Vitamin, 20 ml/L Non‐essential Amino Acid, Sodium Pyruvate and 10% heat‐inactivated FBS (all from Sigma‐Aldrich, Germany)]. The suspension was seeded in a flat‐bottom 48‐well tissue plate (Corning Incorporated, Costar, New York), with 5 × 105 viable cells per well (500 μl). PBMCs were cultured in duplicates either with complete medium alone (unstimulated control) or with one of the following stimulants: 4 μg/ml Resiquimod (R848), 10 μg/ml Zymosan (InvivoGen, France), 10 μg/ml Phytohemagglutinin (PHA‐M), 20 μg/ml Polyinosinic–polycytidylic acid potassium salt (Poly I:C), (Sigma‐Aldrich, Germany), at 37℃, 5% CO2. Cultures were harvested after 48 h and, after centrifugation at 600 g for 5 min, supernatants were stored at −80℃ until analysis.
8
Toll-like Receptor Signaling Assay
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The NF-κB luciferase promoter construct pNF-κB-Luc and the transfection control reporter vector pRL-TK, were purchased from Clontech Laboratories, Inc., (Mountain View, CA), and Promega (Madison, WI), respectively. Wild-type pCMV-TLR1 vector was a gift from Dr Koichi Kuwano (Kurume University School of Medicine, Kurume, Japan). pCMV-MyD88DN expression construct was a gift from Dr. Jason A. Boch (Department of Medicine, Harvard Medical School, Boston, MA). Dominant-negative pZero-hTLR2 expression plasmid, puromycin, blasticidin, zymosan, FSL-1, and Pam3Csk4 were purchased from Invivogen (San Diego, California). IgG2a Isotype control monoclonal antibody was purchased from eBioscience (San Diego, California) and anti-TLR2 (clone TL2.1) monoclonal antibody was purchased from Imgenex (San Diego, California).
9
Measuring IL-6 Responses to TLR Agonists
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DC and myoblasts were harvested and transferred (respectively 50x103 and 10x103 cells/well) to 96-well plates and incubated with LipoPolySaccharide LPS (1 μg/mL), flagellin (10 ng/mL), zymosan (1 μg/mL), CpG-A (10 μg/mL), imiquimod (0.5 μg/mL), poly (I:C) (1 μg/mL), or Pam3CSK4 (100 ng/mL), all from Invivogen, for 16 hr. IL-6 concentration was measured in culture supernatants using a Human IL-6 ELISA MAX Deluxe, according to the manufacturer’s instructions and performing 3 to 4 technical replicates per condition. The results are expressed in picograms per milliliter (pg/mL). Unless otherwise specified in figures or legends, cytokine concentrations in vitro are presented after background subtraction, obtained with cells incubated with vehicle (DMSO or water). In some experiments the TLR9 antagonist ODN TTAGGG (A151) was added 1 hr before addition of stimuli at the concentration of 1 μmol/L.
10
Macrophage Activation and Metabolite Measurement
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