Mice were primed with LPS (0.4 mg kg−1E. coli 0111:B4; #LPS25, Sigma-Aldrich), diluted in PBS for 4 h, via i.p. injection, and acute NLRP3-mediated shock was induced by nigericin (6 mg kg−1, #tlrl-nig, Invivogen) diluted in DMEM via i.p. injection. Survival was followed up to 72 h after nigericin administration.
Lps25
Manufactured by Merck Group
Sourced in United States
The LPS25 is a laboratory equipment product manufactured by Merck Group. It is a sensor designed to measure low pressure levels. The core function of the LPS25 is to detect and report pressure data with high accuracy and reliability.
Automatically generated - may contain errors
Lab products found in correlation
Tlrl nig, by InvivoGen (1 mentions)
Ivis spectrum, by PerkinElmer (1 mentions)
Xenolight rediject inflammation probe, by PerkinElmer (1 mentions)
Luminol sodium salt, by Merck Group (1 mentions)
E coli 0111 b4, by Merck Group (1 mentions)
Living image software, by PerkinElmer (1 mentions)
Ethidium bromide, by Thermo Fisher Scientific (1 mentions)
Cytochalasin b, by MedChemExpress (1 mentions)
Hy b0215, by MedChemExpress (1 mentions)
Mito tempo, by MedChemExpress (1 mentions)
D3308, by Thermo Fisher Scientific (1 mentions)
β sitosterol, by Yuanye Bio-Technology (1 mentions)
Xf96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
C2920, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Sybr green supermix, by Bio-Rad (1 mentions)
Rna extraction kit, by Qiagen (1 mentions)
Cfx connect real time pcr system, by Bio-Rad (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
12 protocols using lps25
1
NLRP3-mediated Shock in Mice
2
In vivo Inflammation Imaging in Mice
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Eight- to twelve-week-old male WT and CD68-POP2 mice had their abdomen shaved under anaesthesia, and were randomly selected for i.p. injection with PBS, LPS (2.5 mg kg−g, E. coli 0111:B4, #LPS25, Sigma-Aldrich) or MSU crystals (3 mg). After 4 h, mice were i.p. injected with XenoLight Rediject Inflammation probe (200 mg kg−1, #760536, PerkinElmer) or luminol (200 mg kg−1) from a 50 mg ml−1 stock solution of Luminol sodium salt (#A4685, Sigma), dissolved in sterile PBS and stored at −20 °C (ref. 68 (link)) and in vivo bioluminescence was captured by imaging (IVIS Spectrum, PerkinElmer) 10 min post injection with a 5-min exposure on anaesthetized mice13 (link)20 (link). Images were quantified with the Living Image software (PerkinElmer). Peritoneal lavages or blood (mandibular bleed) were collected at the indicated time points after PBS, LPS or MSU injection, and cytokine levels were quantified by ELISA.
3
Simulating Pyroptosis in Pulpitis
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mDPC6T cells stimulated with LPS and ATP were utilized to simulate the pyroptosis during the pulpitis pathological process. In detail, mDPC6T cells were first primed with 1 μg/mL LPS (#LPS25, Sigma Aldrich) for 24 h, followed by treatment with 2 mM ATP (#HY‐B2176, MedChemExpress) for 24 h (referred to as LA‐mDPC6T). To inhibit mitochondrial OS, mDPC6T cells were pre‐treated with 5 μM MitoTEMPO (#HY‐112879, MedChemExpress) or 2 μM NAC (#HY‐B0215, MedChemExpress) for 4 h prior to stimulation with LPS and ATP. To inhibit mitochondrial transfer and tunnelling nanotube (TNT) formation, cells were pre‐treated with 1 μM cytochalasin B (HY‐16928, MedChemExpress) for 6 h to interfere polymerization and interaction of actin. For signalling pathway inhibition, cells were pre‐treated with 1 μM NF‐κB inhibitor Bay 11‐7082 (#HY‐13453, MedChemExpress) or 5 μM IKKβ inhibitor ML120B (#HY‐15473, MedChemExpress) for 4 h before experiments. To generate the mBMSCs without the mitochondrial function (ρomBMSCs), cells were cultured in a medium additioned with 1 μg/mL ethidium bromide (15585011, Invitrogen), 100 μg/mL pyruvic acid (HY‐Y0781, MedChemExpress) and 50 μg/mL uridine (HY‐B1449, MedChemExpress) for at least 1 month. For the TNF‐α treatment, the mBMSCs were treated with different concentrations of mouse TNF‐α (#HY‐P7090, MedChemExpress) for 24 h.
4
BBB Disruption and Dextran Extravasation
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BBB damage was induced by IP injection of 3 mg/kg LPS from Escherichia coli (O111:B4, LPS25; Sigma) dissolved in saline. The damage was assessed 24 h after LPS injection using a dextran extravasation assay. The dose and duration of this assay were selected based on Banks et al. (46 (link)). For the dextran extravasation assay, we used 3-kDa TMR-dextran (D3308; Invitrogen) and followed the method described in Devraj et al. (47 ) with one modification—dextran was injected retroorbitally, and therefore blood/brain was collected 5 min after dextran injection. P7C3-A20 (10 mg/kg, IP) was given 12 h after LPS injection.
5
β-sitosterol Modulation of LPS-Induced Responses
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β-sitosterol, with a purity of ≥95% (S24012, Yuan Ye Bio-Technology Co., Ltd., Shanghai, China), was prepared as a stock solution in anhydrous ethanol at a concentration of 40 mmol/mL and stored at −20 °C. The working solutions were further diluted in DMEM to obtain various concentrations: 0.01, 0.1, 1, 5, 10, 20, 30, and 40 µM. LPS from E. coli 0111: B4 (LPS 25, Sigma-Aldrich, St. Louis, MO, USA) was initially dissolved in phosphate buffer solution (PBS) at 5 mg/mL to create the stock solution. This was then diluted to 1 µg/mL in DMEM to serve as the final concentration for subsequent experiments. In a previous study, 1 μg/mL LPS was screened to cause cell damage [25 (link)]. Thus, in subsequent experiments, MAC-T cells were treated with 1 µM β-sitosterol and 1 µg/mL LPS for 24 h. The experiment was divided into four groups, the control group, the β-sitosterol group, the LPS group and the LPS+β-sitosterol group. Each treatment had three independent replicates.
6
LPS-induced Dental Pulp Cell Injury
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DPSCs stimulated with LPS were utilized to simulate the cell injury during the pulpitis pathological process. In detail, DPSCs were primed with 1, 5, and 10 μg/mL LPS (LPS25, Sigma Aldrich) for 24 h, To inhibit mitochondrial transfer and TNT formation, cells were pre‐treated with 1 μM Lat‐B (HY‐16928, MedChemExpress) for 6 h to interfere polymerization and interaction of actin.
7
Extracellular Flux Analysis of Dendritic Cells
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Bone marrow-derived cDC1 stimulated overnight with LPS (O111:B4, Sigma-Aldrich, LPS25), or isolated splenic DCs matured overnight, were seeded prior to analysis at 3 × 105 cells per well on Poly-L-lysine-coated plates (Sigma-Aldrich, P8920), as indicated. Cells were then washed twice and incubated for 1 h in XF assay medium (unbuffered DMEM pH 7.4 with 10 mM glucose, 100 μM sodium palmitate and 2 mM L-glutamine) in a non-CO2 incubator at 37°C as per manufacturer’s instructions (Agilent Technologies). Measurements of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined using an XF-96 Extracellular Flux Analyzer (Agilent Technologies). Serial measurements were obtained under basal conditions and following addition of 1 μM oligomycin (Sigma-Aldrich, 75371), 1.5 μM FCCP (Sigma-Aldrich, C2920) and 100 nM rotenone with 1 μM antimycin A (Sigma-Aldrich, R8875 and A867). For determination of glycolysis, ECAR measurements were obtained under basal conditions.
8
Murine Bone Marrow-Derived Macrophage Isolation and Stimulation
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Tibia, femur and humerus were surgically removed from 8 to 12 weeks old male wild type C57BL6/N mice. Bones were cleaned and surface disinfected with ethanol before the bone marrow was extracted in RPMI-1640. Erythrocytes were lysed in AKC lysis buffer (1 M NH4Cl, 1 M KHCO3, 0.5 M EDTA). Subsequently, cells were subjected to density centrifugation using a Ficoll-Paque gradient and cultured in differentiation medium (DMEM containing 30% L929 supernatant, 20% FBS and 1% penicillin/streptomycin) for 6 days on bacterial plates at 37 °C and 5% CO2. Cells were detached using Versene, counted and seeded in macrophage serum free medium. After overnight incubation, cells were treated with vehicle (PBS), LPS (100 ng/mL, Sigma Aldrich, LPS25) or Dex+LPS (100 ng/mL LPS, 1 µM Dexamethasone, Sigma Aldrich, D2915) for 3 h or as indicated.
9
Transcriptional Response of Immune Cells to PG1 and LPS
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Primary KCs from both wildtype (C57BL/6) and PG1 Tg mice were treated with 10 μg/mL of recombinant purified PG1 in the presence or absence of E. coli O111:B4 LPS (100 ng/mL) (LPS25, Sigma Aldrich) for 12 h. Total RNA was extracted using the Qiagen RNA extraction kit (74104, Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. cDNA was synthesized using Superscript III reverse transcriptase kit (RT006L, Enzynomics, Daejeon, Republic of Korea) according to the manufacturer’s protocol. Real-time quantitative PCR was performed using the SYBR-Green supermix (1725270, Bio-Rad) using CFX connect Real-Time PCR system (Bio-Rad). The following thermocycling conditions were used: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 59 °C for 30 s. The primers used in the present study are listed in Table S2 . Target gene expression was expressed as relative fold change by comparing with the expression of the housekeeping gene (mice GADPH). Relative gene expression was calculated using the 2-ΔΔCT method [57 ].
10
Macrophage COX Activity Modulation
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The effect of treatment with participant plasma and individual metabolites on the total cellular cyclooxygenase (COX) activity of primary macrophage was measured using a commercially available kit (Cayman Chemical, 760151). To induce COX-2 expression, primary macrophages were incubated with 10 ng/mL of lipopolysaccharide (LPS) (Sigma, LPS25) in RPMI and 1xPSG along with 20% participant plasma or in RPMI, 20% FBS, and 1xPSG either alone as a positive control or with isolated metabolites overnight (∼18 h) before measuring total COX activity from cell lysates. A negative control of macrophage in RPMI, 20% FBS, and 1xPSG without LPS was also performed.
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