Granulocyte macrophage colony-stimulating factor (GM-CSF)-derived bone marrow cells (denoted as BMDMs) were prepared as previously described62 (link). In brief, bone marrow cells were grown in RPMI (10-040-CV, Corning cellgro™) supplemented with 10% FBS (TMS-013-B, lot number QVP1404280, Millipore, Billerica, MA or S1620, lot number 221C16, BioWest, Riverside, MO), 1% penicillin–streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, 0.1% β-mercaptoethanol, and 20 ng/mL GM-CSF for 7 days. BMDMs (3 × 106) were seeded in 12-well cell culture plates (3513, Costar) in DMEM (11995-065, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin and infected with A. fumigatus conidia at a multiplicity of infection (MOI) of 20 for 20 h. For priming, BMDMs were incubated with 100 ng/mL of LPS (tlrl-smlps, Invivogen) or 25 μg/mL of curdlan (C7821, Sigma) for 3 h and washed before infecting with A. fumigatus as described above. For polysaccharride transfection, 20 μg curdlan from Alcaligenes faecalis (C7821, Sigma) was resuspended in PBS and mixed with Xfect™ (631318, Takara,) as per the manufacturer’s protocol. The reaction mixture was added to BMDMs in Opti-MEM (31985-070, ThermoFisher Scientific).
Curdlan
Manufactured by Merck Group
Sourced in United States
Curdlan is a lab equipment product manufactured by Merck Group. It is a naturally occurring polysaccharide that functions as a gelling agent and thickener in various applications.
Automatically generated - may contain errors
Lab products found in correlation
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Zymosan, by Merck Group (4 mentions)
E coli lps, by InvivoGen (4 mentions)
Extracellular atp, by Merck Group (4 mentions)
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Functional anti mouse cd3e and cd28 antibodies mabs, by Thermo Fisher Scientific (3 mentions)
Il 1β, by Thermo Fisher Scientific (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Bortezomib, by Selleck Chemicals (2 mentions)
Mannan, by Merck Group (2 mentions)
Zymosan, by InvivoGen (2 mentions)
Zymosan tlrl zyn, by InvivoGen (2 mentions)
Dy215, by R&D Systems (2 mentions)
Dy271, by R&D Systems (2 mentions)
Dy275, by R&D Systems (2 mentions)
Dy208, by R&D Systems (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Scleroglucan, by Merck Group (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Recombinant mouse gm csf, by Thermo Fisher Scientific (2 mentions)
47 protocols using curdlan
1
Fungal Infection of Bone Marrow-Derived Macrophages
2
Intranasal Curdlan Vaccination for P. aeruginosa
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For active immunization, adult female mice were vaccinated intranasally (i.n.) with 20 μL of curdlan (10 mg/mL, Sigma) or purified proteins (25 μg/mouse) plus curdlan (10 mg/mL, Sigma), on days 0, 14, and 21. Mice were challenged at day 35 and were anesthetized with isoflurane or pentobarbital sodium followed by the intratracheal injection of P. aeruginosa XN-1. The lethal dose of P. aeruginosa XN-1 was 1.0 × 107 CFU per mouse. The sublethal dose of P. aeruginosa XN-1 was 1.3 × 106 CFU per mouse.
3
Curdlan-Based Vascular Graft Fabrication
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A solution of 10% curdlan (Sigma Aldrich, St Louis, Mo) or 10% curdlan þ 10% DIP (Sigma Aldrich) mixture film of thickness of 200 mm was made in glass dishes and the hybrid suspensions were sterilized under high-pressure steam. A patch of suitable size heparinized SIS was rolled around a 2-mm diameter quartz rod. The SIS mucosal side was identified and served as the blood-contacting surface. The bilayer SIS with curdlan þ DIP (SCD) or bilayer SIS with curdlan (SC) graft was made by wrapping 2 layers of SIS on the rod with a 10% overlap seam for suture. Size of 20 3 8 mm 10% curdlan or 10% curdlan þ 10% DIP mixture film served as the sandwich filler. Continuous round-trip sutures of 8-0 prolene line were applied to the seam with about 0.5 mm of interval (Figure 1). The suture technique was also applicable to monolayer SIS (Figure 1,A1). The distance of the midpoint of the graft opening was 2 cm. The two ends of the graft retained a single layer structure with a distance 1 mm from the end to match rabbit's native carotid artery (NCA) and 45 oblique angle convenient for bypass anastomosis.
4
Vaccines against Pseudomonas aeruginosa
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FpvA and FpvA-KLH peptides were resuspended in 125 mM NaOH (Fisher Scientific). The unconjugated FpvA peptide vaccine was prepared using a mix of 35 μg of each FpvA peptide in PBS. The FpvA-KLH conjugate vaccine dose consisted of a mix of 35 μg of each FpvA-KLH peptide in PBS. The whole-cell vaccine (WCV) was prepared using P. aeruginosa PAO1 “Vasil.” The strain was grown as described above. Swabbed bacteria were resuspended in Phosphate Buffered Saline (PBS), and heat-killed at 60°C for 1 h. The efficiency of heat-killing was verified by plating on PIA. Each WCV dose consisted of 4–5 × 107 heat-killed colony-forming unit (CFU) of P. aeruginosa in PBS. All vaccines were prepared in a final volume of 20 μl and contained 100 μg of curdlan (Sigma Aldrich) as an adjuvant unless otherwise specified. The curdlan-only control consisted of 100 μg of curdlan in PBS.
5
Callose Analogues Characterization
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Avicel PH-101 (cellulose)
and the (1,3)-β-d -glucan Curdlan were purchased from
Sigma Aldrich. The (1,3)-β-d -glucan Pachyman was purchased
from Biosupplies Australia (www.biosupplies.com.au ). Pachyman and Curdlan were used as
callose commercial analogues. The ionic liquid 1-ethyl-3-methyl-imidazolium
acetate [emim][OAc] (97% purity) was purchased from Sigma Aldrich.
The cationic dye methylene blue (MB) was purchased from Sigma Aldrich
and dissolved in Milli-Q water (Merck Millipore, Darmstadt, HE, Germany).
and the (1,3)-β-
Sigma Aldrich. The (1,3)-β-
from Biosupplies Australia (
callose commercial analogues. The ionic liquid 1-ethyl-3-methyl-imidazolium
acetate [emim][OAc] (97% purity) was purchased from Sigma Aldrich.
The cationic dye methylene blue (MB) was purchased from Sigma Aldrich
and dissolved in Milli-Q water (Merck Millipore, Darmstadt, HE, Germany).
6
Curdlan Characterization and Endotoxin Detection
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Curdlan was purchased from Sigma-Aldrich (St. Louis, MI, USA), and the viscosity-average molecular weight, obtained using the gel permeation chromatography (GPC) method, was 7.9 × 105 g/mol. Ethylene glycol diglycidyl ether (EGDGE), NaOH (99.99%), hydrochloric acid, and deuteroxide (D2O, 99.8% D), Tris•HCl buffer (pH 8.0) were purchased from J&K (Shanghai, China) and used without further treatment. Chromogenic end-point Tachypleus Amebocyte Lysate Kit (CE TAL Kit) was purchased from Xiamen Bioendo Technology Co., Ltd. (Fujian, China).
7
Dectin-1-mediated Murine Dendritic Cell Activation
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C57BL/6 and Balb/c mice were purchased from the Jackson Laboratory. Dectin-1 -/-mice were provided by G. Brown (University of Aberdeen, Aberdeen, Scotland). Mice were bred and maintained in pathogen-free facilities at the First Hospital Animal Center of Jilin University. 6 to 8-week-old mice were used for the experiments. All animal experimental procedures were reviewed and approved by the Animal Ethical Committee of First Hospital of Jilin University.
RPMI-1640 medium, penicillin and streptomycin were purchased from Invitrogen. Fetal bovine serum was purchased from Hyclone. Recombinant murine granulocytemacrophage colony-stimulating factor (GM-CSF), IL-4, IL-1β and tumor necrosis factor-alpha (TNF-α) were purchased from Peprotech. Recombinant human transforming growth factor-beta (TGF-β) was purchased from R&D Systems. Functional anti-mouse CD3e and CD28 antibodies (mAbs) were purchased from eBioscience. Curdlan was purchased from Sigma-Aldrich. Lipopolysaccharide (LPS) and poly I:C were purchased from Sigma-Aldrich and Invivogen respectively. Syk inhibitor Piceatannol and Raf-1 kinase inhibitor GW5074 were purchased from Calbiochem. NF-κB inhibitor Bortezomib was purchased from Selleckchem. NF-κB inhibitor (CAS 213546-53-3) was purchased from Santa Cruz, and JSH-23 (a NF-κB inhibitor) was purchased from MCE.
RPMI-1640 medium, penicillin and streptomycin were purchased from Invitrogen. Fetal bovine serum was purchased from Hyclone. Recombinant murine granulocytemacrophage colony-stimulating factor (GM-CSF), IL-4, IL-1β and tumor necrosis factor-alpha (TNF-α) were purchased from Peprotech. Recombinant human transforming growth factor-beta (TGF-β) was purchased from R&D Systems. Functional anti-mouse CD3e and CD28 antibodies (mAbs) were purchased from eBioscience. Curdlan was purchased from Sigma-Aldrich. Lipopolysaccharide (LPS) and poly I:C were purchased from Sigma-Aldrich and Invivogen respectively. Syk inhibitor Piceatannol and Raf-1 kinase inhibitor GW5074 were purchased from Calbiochem. NF-κB inhibitor Bortezomib was purchased from Selleckchem. NF-κB inhibitor (CAS 213546-53-3) was purchased from Santa Cruz, and JSH-23 (a NF-κB inhibitor) was purchased from MCE.
8
Rapid (1,3)-β-D-glucan Detection
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Restriction enzymes Xba I, Hind III and T4 ligase were purchased from Fermentas (Thermo Fisher Scientific, Germany). DNA markers and protein markers were purchased from LabLead Co., Ltd (Beijing, China). PCR mix was purchased from JiErDun Co., Ltd (Shanghai, China). Salmon sperm DNA, hemin, and TMB were purchased from TaiTianHe Co., Ltd (Jinan, China). A turbidimetric (1,3)-β-D-glucan test kit was purchased from Bokang Marine Biological Company (Zhanjiang, China). Barley glucan, curdlan, mannan, endotoxin, dextran, and (1,3)-β-D-glucanase were sourced from Sigma–Aldrich (St. Louis, MO). ssDNA libraries and primers were synthesized by Sangon Biotech Co., Ltd (Shanghai China).
9
Quantifying β-1,3-D-glucan in Mycelial Tissue
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The β-1,3-D-glucan content was determined by aniline blue staining according to previously described methods [32 (link)]. In short, fluorescence readings were obtained using a Tecan Infinite M200 fluorometer with excitation at 405 nm and emission at 460 nm. The absorbance was measured at 650 nm with a Vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan). For normalization of the values, a standard curve was created using curdlan, a β-1,3-D-glucan analog (Sigma, St. Louis, MO, USA). The values are expressed as the percentage changes in relative fluorescence units per milligram of mycelial tissue using WT as the control.
10
Endotoxin-free β-glucan preparation
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Macrogard was kindly provided by Biotec Pharmacon ASA (Tromsø, Norway) and Curdlan was purchased from Sigma (Bornem, Belgium). All material coming into contact with the β-glucans was made pyrogen-free. To this end, clean glassware and material was heated at 250°C for 4 h. β-glucan solutions were made in LAL reagent water (Catalog number W50-100; Lonza, Basel, Switzerland). Both types of β-glucan were solubilized via sonication. Quantification of endotoxin concentrations present in the β glucan preparations, was done by the Chromogenic Limulus Amebocyte Lysate (LAL) Test (Catalog number 50-647U, Lonza) and were 0.0362 EU/μg MG and 4.22 EU/μg CL.
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