O5 b55 lps

Manufactured by InvivoGen

O5:B55 LPS is a bacterial lipopolysaccharide (LPS) product. LPS is a key component of the outer membrane of Gram-negative bacteria and is known to stimulate the immune system. The O5:B55 LPS is purified from Escherichia coli O55:B5.

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Lab products found in correlation

Myxothiazol, by Merck Group (2 mentions) Dimethyl malonate, by Merck Group (2 mentions) Oligomycin, by Merck Group (2 mentions) Retroscript first strand synthesis kit, by Thermo Fisher Scientific (2 mentions) Piericidin a, by Cayman Chemical (2 mentions) Iq sybr green supermix, by Bio-Rad (2 mentions) Adenosine triphosphate (atp), by Merck Group (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions) Tnf α dy410, by R&D Systems (1 mentions) O5 b55 lps, by Merck Group (1 mentions)

5 protocols using o5 b55 lps

Quantitative PCR of LPS-treated BMDMs

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Quantitative PCR reverse transcription (qRT–PCR) was performed on BMDMs treated with 100 ng ml^–1 ultrapure O5:B55 LPS (Invivogen, catalog no. tlrl-pb5lps) for 4 h. Before stimulation, BMDMs were treated for 30 min with piericidin A (Cayman Chemical, catalog no. 15379), myxothiazol (Sigma, catalog no. T5580), FCCP (Sigma, catalog no. C2920), oligomycin (Sigma, catalog no. 75351) or dimethyl malonate (Sigma, catalog no. 136441), as indicated in figures and figure legends.
RNA was extracted from peritoneal macrophages or BMDMs using the Omega E.Z.N.A. RNA Isolation Kit (Omega Biologicals, catalog no. R6834-02). RNA was quantified using a Nanodrop 2000 UV-visible spectrophotometer, and 300 ng of RNA was reverse transcribed using RETROscript first-strand synthesis kit (ThermoFisher, catalog no. AM17-10). Real-time PCR was performed on a BioRadCFX using iQ SYBR green Supermix (Bio-Rad, catalog no. 1708880). The following primers were used: IL-1β (forward 5′- TGGCAACTGTTCCTG-3′; reverse 3′-GGAAGCAGCCCTTCATCTTT-5′); NDI1 (forward 5′-GCCGAAGAAGTCCAAATTCAC-3′; reverse 3′- CGACAGCCGTTCTCAGAT-5′); b-Actin (forward 5′-CTAAGGCCAACCGTGAAAA-3′; reverse 3′-ACCAGAGGCATACAGGGACA-5′). Fold changes in gene expression relative to untreated control were calculated by the ΔΔC_t method using mouse actin as an endogenous control for mRNA expression.

Billingham L.K., Stoolman J.S., Vasan K., Rodriguez A.E., Poor T.A., Szibor M., Jacobs H.T., Reczek C.R., Rashidi A., Zhang P., Miska J, & Chandel N.S. (2022). Mitochondrial electron transport chain is necessary for NLRP3 inflammasome activation. Nature Immunology, 23(5), 692-704.

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Inflammasome Activation and Cytokine Release

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BMDMs were stimulated with O5:B55 LPS (Invivogen) for 6 hours with addition of 5mM ATP (Sigma) for 30 minutes to activate the inflammasome. Cell supernatant was collected and analysed by ELISA according to manufacturers instructions for IL-1β (R&D Duoset DY401–05, R&D Quantikine MLB00C), TNFα (R&D Quantikine MTA00B), and IL-18 ELISA (MBL 7625). Caspase-1 activation was by western blot analysis using the anti-caspase 1 from Adipogen (AG-20B-0042), and the control was anti-vinculin (Fisher NC1391571). Secondary antibodies were from LicCOR and the gel was imaged on LiCOR. Densitometry was determined using SoftMax pro software. The ratio of cleaved caspase to vinculin in serine-deprived cells was expessed as a percentage of the cleaved caspase-to-vinculin ratio in cells stimulated with serine-replete media.

Rodriguez A.E., Ducker G.S., Billingham L.K., Martinez C.A., Mainolfi N., Suri V., Friedman A., Manfredi M.G., Weinberg S.E., Rabinowitz J.D, & Chandel N.S. (2019). Serine metabolism supports macrophage IL-1β production.. Cell metabolism, 29(4), 1003-1011.e4.

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qRT-PCR Analysis of LPS-Induced Gene Expression

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qRT-PCR was performed on BMDMs treated with 100ng/mL ultrapure O5:B55 LPS (Invivogen, tlrl-pb5lps) for 4 hours. Prior to stimulation, BMDMs were treated for 30 minutes with piericidin A (Cayman Chemical, 15379), myxothiazol (Sigma, T5580), Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP; Sigma, C2920) oligomycin (Sigma, 75351), or dimethyl malonate (Sigma, 136441), as indicated in figures and figure legends.
RNA was extracted from peritoneal macrophages or BMDMs using the Omega E.Z.N.A. RNA Isolation Kit (Omega Biologicals, R6834-02). RNA was quantified using a Nanodrop 2000 UV-visible spectrophotometer, and 300ng of RNA was reverse transcribed using RETROscript first-strand synthesis kit (Thermo Fisher, AM17-10). Real-time PCR was performed on a BioRadCFX using iQ SYBR green Supermix (Bio-Rad, 1708880). The following primers were used: IL-1β (Fwd 5’- TGGCAACTGTTCCTG-3’ Rev 3’-GGAAGCAGCCCTTCATCTTT-5’); NDI1 (Fwd 5’-GCCGAAGAAGTCCAAATTCAC-3’ Rev 3’- CGACAGCCGTTCTCAGAT-5’); b-Actin (Fwd 5’-CTAAGGCCAACCGTGAAAA-3’ Rev 3’-ACCAGAGGCATACAGGGACA-5’). Fold changes in gene expression relative to untreated control were calculated by the ΔΔC_t method using mouse actin as an endogenous control for mRNA expression.

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LPS-Induced Sepsis Model in Mice

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LPS-induced sepsis model in mice was approved by Northwestern University Institutional Animal Care and Use Committee. For short term LPS-induced cytokine quantification, C57/Bl6 mice (aged 10–12 weeks) were treated intraperitoneally with or without 1400W (10mg/kg) for 2hrs prior to intraperitoneal injection with ultrapure O5:B55 LPS from Invivogen (15mg/kg) for 4hrs. Whole blood samples were harvested via cardiac puncture after mice were euthanized. Cytokine production in serum from whole blood was measured using the Mouse IL-1β (DY401), IL-6 (DY406), and TNFα (DY410) ELISAs from R&D. For survival studies, Crude O5:B55 LPS (Sigma) was administrated intraperitoneally at a sub-lethal dose of 15 mg/kg and mice were monitored over 72hrs, every 2–4hrs for survival and signs of deterioration to determine humane endpoints (Shrum et al., 2014 (link)).

De Jesus A., Keyhani-Nejad F., Pusec C.M., Goodman L., Geier J.A., Stoolman J.S., Stanczyk P.J., Nguyen T.A., Xu K., Suresh K.V., Chen Y., Rodriguez A.E., Shapiro J.S., Chang H.C., Chen C., Shah K.P., Ben-Sahra I., Layden B.T., Chandel N.S., Weinberg S.E, & Ardehali H. (2022). Hexokinase 1 cellular localization regulates the metabolic fate of glucose. Molecular cell, 82(7), 1261-1277.e9.

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LPS-Induced Inflammasome Activation in BMDMs

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BMDMs were stimulated with O5:B55 LPS (Invivogen) for 6hrs with addition of 2mM ATP (Sigma) for 30min to activate the cleavage of pro-IL-1β (35kDa) to cleaved-IL-1β (17kDa). Cell supernatant was collected and analyzed by ELISA according to manufacturer’s instructions for IL-1β (DY401), IL-6 (DY406), and TNFα (DY410). Briefly, highly absorbent 96 well plates were used to bind the capture antibody (IL-6, TNFα, or IL-1β) which was diluted in PBS and left overnight to adhere to the 96 well plate. The next day, the plate was washed 3 times with wash buffer (PBS, 0.05% Tween20). Samples were then diluted in reagent dilution buffer (PBS, 1% BSA, 0.5X Azide, stored at 4°C), added to the plate and incubated for 2hrs. The plate was washed 3× with wash buffer and incubated for 2hrs with detection antibody, which was diluted in reagent dilution buffer. The plate was washed 3× with wash buffer and the fluorescence reaction was performed using Streptavidin-HRP-based reaction provided by the ELISA kit.

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