MSU crystals were prepared as described by Denko and Whitehouse [9 (link)]. Briefly, 4 g uric acid was dissolved in 800 ml of deionized water, heated to 60 °C, adjusted to pH 8.9 with 0.5 N NaOH, and crystallized overnight at room temperature. MSU crystals were recovered by centrifugation, washed with distilled water and dried at 40 °C for 24 h. Crystal shape and birefringence were assessed by compensated polarized light microscopy. MSU crystals were milled and then sterilized by heating at 180 °C for 2 h before each experiment. We measured <0.015 EU/ml endotoxin in MSU crystal preparations using a Limulus amebocyte lysate assay (Sigma-Aldrich, St Louis, MO, USA).
Limulus amebocyte lysate assay
Manufactured by Merck Group
Sourced in United States
Limulus amebocyte lysate (LAL) assay is a lab equipment product used for the detection and quantification of endotoxins, also known as lipopolysaccharides (LPS). The assay utilizes the clotting reaction of the LAL, which is derived from the blood cells of the horseshoe crab (Limulus polyphemus). The presence of endotoxins in a sample triggers a cascade of enzymatic reactions in the LAL, leading to the formation of a detectable gel or color change. The LAL assay is a widely used method for ensuring the safety and quality of pharmaceutical, medical, and other products by verifying the absence of endotoxin contamination.
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Lab products found in correlation
Hank s balanced salt solution, by Merck Group (4 mentions)
Bl21 de3 plyse, by Thermo Fisher Scientific (1 mentions)
Sulforaphane, by Merck Group (1 mentions)
7 aminoactinomycin d, by BD (1 mentions)
Annexin 5 fitc kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometry, by Thermo Fisher Scientific (1 mentions)
Imiquimod r837, by InvivoGen (1 mentions)
Dsrna poly 1 c, by InvivoGen (1 mentions)
E toxate kit, by Merck Group (1 mentions)
15 protocols using limulus amebocyte lysate assay
1
Monosodium Urate Crystal Preparation
2
Polyclonal IgG Purification and Characterization
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All polyclonal IgG was purified by protein G–Sepharose chromatography (Pierce, UK), passed through Detoxi-Gel™ Endotoxin removing columns (Pierce, UK) and subsequently determined to be endotoxin free (<0.125 Endotoxin units/mL) by the Limulus amebocyte lysate assay (Sigma, Gillingham, UK). The polyclonal APS-IgG was purified from stored serum samples that were confirmed to have aCL and anti-β2GPI activity. The aCL and anti-β2GPI activity of IgG was then measured as described previously16 (link) using international calibrators in G phospholipid units (GPLU, from APL Diagnostics, Galveston, TX, USA) for the CL assay and an in-house standard of a patient with positive aPL (but no APS) with known anti-β2GPI binding for the anti-β2GPI assay [results expressed as standard units (SU)]. Pooled IgG was obtained by combining an equal concentration of IgG with similar aPL binding from five individual samples in the VT+/PM−, VT−/PM+ and HC groups shown in TableI . Two different batches, each consisting of overlapping IgG from the three comparator groups, of pooled IgG were used in these experiments.
3
Preparation and Evaluation of Advanced Glycation End-Products
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AGEs were prepared according to the reported methods in previous literature (14 (link)). For this purpose, 50 mg/ml bovine serum albumin (BSA) was incubated with glucose generated in non-enzymtic glyction in the dark at 37 °C for 16 weeks. Under the same condition, glycoprival was incubated and acted as the control group. The unincorporated sugars were removed by dialyzing against PBS after incubation. Limulus amebocyte lysate assay (Sigma, St. Louis, MO, USA) was used to evaluate the level of endotoxin.
4
Recombinant HMGB1 Protein Production
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For the recombinant HMGB1 protein, six-His-tagged recombinant human WT HMGB1, HMGB1 boxes A (aa 1-79) and B (aa 88-162), and acidic tail-deleted HMGB1 (ΔC-HMGB1, aa 1-185) were subcloned into pRSET B plasmid and produced in Escherichia coli BL21 (DE3) pLysE (Invitrogen) (4 (link), 16 (link)). A six-His-tagged HMGB1 (ΔN-HMGB1, aa 11-215), a form of HMGB1 with pro-inflammatory potential (29 (link)), was subcloned into pRSET B plasmid and produced in E. coli BL21 (DE3) pLysE. One mM DTT was added during protein purification and preservation. Endotoxin was removed using an LPS-binding column (Thermo Fisher Scientific, Inc.) or detergent-phase separation using Triton X-114 (30 (link)). LPS concentrations were less than 0.1 EU/μg protein, as determined using the limulus amebocyte lysate assay (Sigma). In addition, HMGB1 produced in NS0 mouse myeloma cell line (Euk-HMGB1, R&D Systems) was used to confirm the study.
5
Preparation and Characterization of Aqueous Organic Dust Extract
6
Preparation and Characterization of Swine Dust Extracts
7
Swine Confinement Dust ODE Preparation
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Aqueous ODE was prepared as previously described [29 (link)]. Briefly, settled dust was collected from horizontal surfaces (~3 feet above floor level) of swine confinement feeding operations, with knowledge and permission granted by the swine confinement owners. These operations are located in Colfax County, Nebraska (population density approximates 25 people per square mile). Dust (1 gm) was incubated in sterile Hank’s Balanced Salt Solution (10 ml; Sigma, St. Louis, MO) at room temperature for 1 hour, centrifuged for 60 min at 2000 x g, and the final supernatant was filter sterilized (0.22 μm), a process that also removes coarse particles. Endotoxin concentrations in 100% ODE ranged from 1240–1400 EU/ml as determined using the limulus amebocyte lysate assay (Sigma). Muramic acid levels were previously determined by mass spectrometry to be approximately 70 ng/mg [14 (link)]; muramic acid is a molecular component of bacterial cell wall peptidoglycans. Stock ODE was diluted to a final concentration (vol/vol) of 0.5% for cell culture studies and 12.5% for animal studies in sterile phosphate buffered saline (PBS; pH: 7.4; diluent). ODE 12.5% has been previously shown to elicit optimal experimental outcomes in mice and is well tolerated [30 (link)].
8
Monosodium Urate Crystal Characterization
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MSU crystals were prepared as we previously reported (23 (link)) and then assessed using compensated polarized light microscopy. Endotoxin was present at <0.015 EU/mL in the MSU crystal preparations, as determined using the Limulus amebocyte lysate assay (Sigma-Aldrich, St Louis, MO, USA). Before each experiment, the MSU crystals were milled and then sterilized for 2 h.
9
Evaluation of Wine Extract's Antioxidant Effects
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To explore the effect of wine extract in counteracting oxidative stress, increasing concentrations (from 200 to 800 μg/mL of total polyphenols) of wine extract were added to TPH-1 cells and cardiomyocytes for 6 h, before the addition of TBHP. As a positive control, THP-1 cells and cardiomyocytes were preincubated in some experiments with increasing concentrations (1–10 μM) of the isothiocyanate sulforaphane (Sigma Chemical Co, St. Louis, MI, USA) known to activate the Keap1-Nrf2 signaling pathway [42 (link)]. Early apoptosis and cell viability was determined using the Annexin V-FITC Kit (Bender MedSystems GmbH, Vienna, Austria) and 7-amino-actinomycin D (BD Biosciences, Buccinasco, Italy) by flow cytometry. Endotoxin contamination of cell cultures was routinely excluded with the chromogenic Limulus amebocyte lysate assay (Sigma-Aldrich, Milan, Italy).
10
Swine Dust Extract Inflammation Protocol
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Aqueous organic dust extract (ODE) was prepared from settled dust collected from horizontal surfaces 3 ft. above the floor in swine confinement feeding operations. Extracts were batch prepared and filter (0.22 μm) sterilized as previously described [21 (link), 22 (link)]. Stock ODE was diluted to a concentration of 12.5% (vol/vol) in sterile phosphate buffered saline (PBS, pH 7.4, diluent), a concentration previously shown to elicit optimal lung inflammation in mice [21 (link), 22 (link)]. ODE 12.5% contains approximately 3–4 mg/ml total protein as measured by Nanodrop spectrophotometry (NanoDrop Technologies, Wilmington, DE) and endotoxin levels range from 22.1 to 91.1 EU/ml as measured by the limulus amebocyte lysate assay using manufacturer instructions (Sigma). Specific microbial biomarkers inferred from shotgun metagenomics of DNA pyrosequencing reads of the dust samples have been previously detailed [23 (link)].
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