Trained immunity was induced in an in vitro model, as described previously [11 (link)]. Briefly, monocytes were obtained as described above and plated in flat-bottom 96-well plates, with 105 cells per well, and in some experiments, pre-incubated with IL-38 (3-152, 90 ng/mL; R&D systems, Minneapolis, MN, USA), IL-36RA (400 ng/mL; PeproTech, London, UK), or an InSolution NF-κB activation inhibitor (100 nM; Calbiochem, San Diege, CA, USA) for 1 h at 37 °C, or with supplemented RPMI, as a negative control. Subsequently, cells were trained for 24 h at 37 °C using Bacillus Calmette-Guerin (BCG; 2 μg/mL; SSI, Copenhagen, Denmark), β-1,3-(D)-glucan (2 μg/mL; kindly provided by Professor David Willems, Johnson City, USA), and IL-36γ (50 ng/mL; PeproTECH) using supplemented RPMI as a negative control. After 24 h, cells were washed with warm PBS and incubated in supplemented RPMI with 10% human serum for five days. On day six, cells were restimulated for 24 h with lipopolysaccharide (LPS) (10 ng/mL) from E. coli serotype O55:B5 (Sigma-Aldrich, Darmstadt, Germany). Supernatants were stored at −20 °C until further analysis.
E coli serotype o55 b5
Manufactured by Merck Group
Sourced in United States
E. coli serotype O55:B5 is a strain of Escherichia coli bacteria. It is a laboratory tool used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Complete rpmi 1640, by Merck Group (1 mentions)
Dextrose, by Pfizer (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Psb1115, by Bio-Techne (1 mentions)
8 protocols using e coli serotype o55 b5
1
Trained Immunity Induction Protocol
2
Dendritic Cell Activation by CM-272
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5x105 DCs were exposed to 0.05-1μM CM-272 for 24 hours in a 48-well plate in 500μL complete RPMI-1640 (Sigma-Aldrich) and cultured for an additional 24 hours with/without 1μg/mL lipopolysaccharide (E. coli serotype O55:B5; Sigma-Aldrich). DCs were collected for flow cytometry and IL-12p70 was measured in culture supernatants. DCs pulsed with 10μg/mL OVA257-264 were co-cultured at 1:10 ratio with OT-I T cells for 72 hours before IFN-γ measurement. Unstimulated and CD3/CD28-stimulated (ThermoFisher, Waltham, Massachusetts) T cells served as negative and positive controls, respectively.
3
LPS-induced Inflammation Model in Mice
4
Hyperdynamic Endotoxemic Shock Management
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A description of the model and methodology has been published previously [24 ]. Briefly, hyperdynamic endotoxemic shock was induced by administration of an escalating dose of lipopolysaccharide (LPS; E coli serotype O55:B5; Sigma-Aldrich, St Louis, USA) up to 4 μg/kg/h over 4 h (total LPS dose 11.25 μg/kg). Adequate endotoxemia was confirmed by the occurrence of systemic hypotension with a mean arterial pressure (MAP) less than 60 mmHg after 3 h of endotoxin infusion. In the final hour of endotoxemia animals received either fluid resuscitation with 40 mLs/kg of 0.9% saline over an hour (FR; n = 8) or commenced protocolised vasopressor support (NFR; n = 8). Noradrenaline was started 60 μg/mL in 5% dextrose (Hospira, Lake Forest, IL, USA) to maintain a MAP between 60 and 65 mmHg. If noradrenaline reached a predetermined 20 μg/min, vasopressin (PPC, Richmond Hill, ON, Canada) was commenced at 0.8 units/h and increased to a maximum of 1.6 units/h if hypotension persisted. The administration protocol was the same for both groups. Post-fluid resuscitation, both groups were monitored for 12 h after the end of endotoxin infusion.
5
Endotoxin-induced cytokine release assay
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After approval from the local ethics committee of the Radboud university medical center (CMO 2010/10), lithium heparin-anticoagulated blood was obtained from 8 healthy volunteers who provided written informed consent. Blood was diluted 5 times in culture medium (RPMI [Invitrogen, Carlsbad, California, USA] supplemented with 10 μg/mL gentamicin, 10 mM Glutamax and 10 mM pyruvate) and incubated in 48-well plates in the presence of 1 or 10 μM PSB1115 (Tocris BioScience, Abingdon, UK) or vehicle (DMSO, final concentration of 0.1% in all experimental conditions) for 30 min at 37 °C and 5% CO2. Hereafter, 10 μM 5′-N-Ethylcarboxamidoadenosine (NECA) or vehicle (DMSO) was added and cultures were again incubated for 30 min. Subsequently, 10 ng/mL endotoxin (E. coli, serotype O55:B5, Sigma Aldrich) or vehicle (RPMI) was added and cultures were incubated for 24 h, after which plates were centrifuged for 8 min at 1400 RPM at room temperature and supernatant collected and stored at −80 °C until analysis. Cytokine concentrations were measured using ELISA according to the manufacturer's instructions (Human Duoset, R&D systems).
6
Chicken Immune Response to LPS
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The SPF male Leghorn chickens (21 days old) were purchased from Beijing Boehringer Ingelheim Witon Biotechnology Co., Ltd., Beijing China; the lipopolysaccharide (LPS) was E. coli serotype O55:B5, purchased from Sigma-Aldrich Chemical Co., St. Louis, MI, USA; the Sihuang Zhili granules were from Baoding Jizhong Pharmaceutical Co., Ltd., Baoding, China.
7
Chronic LPS-Induced COPD Model
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PI was induced by the bi-weekly intratracheal administration of lipopolysaccharides (LPS) 0.4 mg/mL, diluted in NaCl 0.9% for 4 weeks (1 mL/kg of body weight; E. coli, serotype O55:B5; Sigma-Aldrich Chemical Co.™, St Louis, MO, USA). This model is reported to induce airway remodeling, with similarities to those observed in COPD patients [25 (link),26 (link)].
During the last 14 days of the PI protocol, animals were exposed to a either chronic normoxia (N groups) or normobaric hypoxia (CH groups, FiO2: 10%). The hypoxia chamber was opened for 45–60 min per day to weigh animals and perform LPS administration and nursing, as previously described [27 (link)].
During the last 14 days of the PI protocol, animals were exposed to a either chronic normoxia (N groups) or normobaric hypoxia (CH groups, FiO2: 10%). The hypoxia chamber was opened for 45–60 min per day to weigh animals and perform LPS administration and nursing, as previously described [27 (link)].
8
Chronic Pulmonary Inflammation via LPS
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A chronic pulmonary inflammation was induced by bi-weekly lipopolysaccharides (LPS) intracheal administration to the animals at 1 mL/kg of body weight (E.coli, serotype O55:B5; Sigma-Aldrich Chemical Co.™, St Louis, Missouri, USA in NaCl 0.9% at 0.4 mg/mL) for 4 weeks as previously described32 (link).
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