E coli k12 lps

Manufactured by InvivoGen

Sourced in United States

E. coli K12 LPS is a purified lipopolysaccharide (LPS) derived from the K12 strain of Escherichia coli. LPS is a major component of the outer membrane of Gram-negative bacteria and plays a crucial role in their structural integrity and cellular processes.

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Lab products found in correlation

Pam3csk4, by InvivoGen (3 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Iscove s modified dulbecco s media, by Thermo Fisher Scientific (1 mentions) C57bl 6j, by Jackson ImmunoResearch (1 mentions) Nigericin, by InvivoGen (1 mentions) Mouse anti ha 11 clone 16b12, by BioLegend (1 mentions) Phorbol myristate acetate (pma), by Santa Cruz Biotechnology (1 mentions) Doxycycline hyclate, by Merck Group (1 mentions) Hrp coupled sheep anti mouse igg, by GE Healthcare (1 mentions) Tak 242, by Merck Group (1 mentions) Ly294002, by Cell Signaling Technology (1 mentions) Anti phospho sapk jnk, by Cell Signaling Technology (1 mentions) Odn2006, by InvivoGen (1 mentions) Anti phospho akt ser473, by Cell Signaling Technology (1 mentions) Anti phospho p38 mapk, by Cell Signaling Technology (1 mentions) Quanti blue, by InvivoGen (1 mentions) Marimastat, by Merck Group (1 mentions) Anti pan akt, by Cell Signaling Technology (1 mentions) Anti nf κb p65, by Cell Signaling Technology (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)

Close spelling variants of this product

Lipopolysaccharide (LPS) from E. coli K12 LPS from E. coli K12 strain (LPS-EK) E. coli K12 lipopolysaccharide (LPS) Ultrapure LPS from E. coli K12 E. coli K12 ultra-pure LPS Ultrapure LPS (E. coli K12) LPS from E. coli K12 E. coli K12 E. coli LPS

14 protocols using e coli k12 lps

Inducing Pro-inflammatory Responses in Cell Cultures

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Escherichia coli K12 lipopolysaccharide (LPS; 0.1, 1.0, and 10.0 µg/ml; InvivoGen, San Diego, CA) and Pam3CSK4 (0.1, 1.0, and 10.0 µg/ml; InvivoGen, San Diego, CA) were used as agonists to induce pro-inflammatory responses in single cell cultures and multi-cell cultures. Weight per volume stock solutions were prepared in pyrogen-free 0.01 M sodium phosphate with 0.140 M NaCl, pH 7.2 (PBS) containing 4.0 + 0.7 SEM (n = 3) pg/ml endotoxin (QCL-1000, Lonza Walkersville, Inc., Walkersville, MD). Stock solutions were then diluted in LGM-3 before use.
10.0 µg/ml E. coli K12 LPS (InvivoGen, San Diego, CA) and 10.0 µg/ml Pam3CSK4 (InvivoGen, San Diego, CA) were selected as optimum doses for each agonist and used to induce pro-inflammatory events in both the multi-cell computational models and multi-cell cultures.

Fischer C.L., Bates A.M., Lanzel E.A., Guthmiller J.M., Johnson G.K., Singh N.K., Kumar A., Vidva R., Abbasi T., Vali S., Xie X.J., Zeng E, & Brogden K.A. (2019). Computational Models Accurately Predict Multi-Cell Biomarker Profiles in Inflammation and Cancer. Scientific Reports, 9, 10877.

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Intracellular Cytokine Analysis of Blood and BAL Samples

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Directly after collection, blood and BAL samples were added to an equal volume (100 μL/tube) to IMDM (Iscove’s Modified Dulbecco’s Media, Thermo Fisher Scientific, Waltham, MA, USA) with or without 200 ng/mL of Escherichia coli LPS (E. coli K12 LPS, Invivogen, San Diego, CA, USA), then incubated for 4 h at 37 °C and 5% CO₂ [23 (link)]. Samples drawn to measure the intracellular cytokine synthesis of TNF-α were incubated with a Golgi inhibitor (GolgiStop™ 2 μM, Becton Dickinson, San Jose, CA, USA) for 4 h at 37 °C and 5% CO₂.

Bendib I., Beldi-Ferchiou A., Schlemmer F., Maitre B., Surenaud M., Hüe S., Carteaux G., Razazi K., Lelièvre J.D., Lévy Y., Mekontso Dessap A., Godot V, & de Prost N. (2021). Functional Ex Vivo Testing of Alveolar Monocytes in Patients with Pneumonia-Related ARDS. Cells, 10(12), 3546.

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Bone Marrow-Derived Dendritic Cell Generation

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Bone marrow-derived DCs were generated from 6–8 week old female C57BL/6J (Jackson Laboratories), Ap1ar^−/− (Maritzen et al., 2012 ), Samhd1^−/− (Rehwinkel et al., 2013 (link)), Myd88^−/−, Myd88^−/−/Ticam^−/−, Irak2^−/−, Irak4^−/− mice. All stimulations were performed using ultra-pure E. coli K12 LPS (Invivogen) at 100 ng/mL. For shRNA knockdowns, high-titer lentiviruses expressing shRNAs were used to infect bone marrow cells as previously described (Chevrier et al., 2011 (link)).

Mertins P., Przybylski D., Yosef N., Qiao J., Clauser K., Raychowdhury R., Eisenhaure T.M., Maritzen T., Haucke V., Satoh T., Akira S., Carr S.A., Regev A., Hacohen N, & Chevrier N. (2017). An Integrative Framework Reveals Signaling-to-Transcription Events in Toll-Like Receptor Signaling. Cell reports, 19(13), 2853-2866.

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Reagents and Antibodies for Inflammasome Assays

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Doxycycline hyclate was purchased from Sigma-Aldrich; E. coli K12 LPS, nigericin, and poly(dA:dT) were obtained from InvivoGen. PMA was purchased from Santa Cruz Biotechnology, Inc.; TAK-242 was obtained from EMD Millipore; and z-YVAD-cmk was purchased from Bio-Techne. Mouse anti-HA.11 (clone 16B12) was acquired from BioLegend, mouse anti-EGFP (clone JL-8) was purchased from Takara Bio Inc., mouse anti–IL-1β (clone 3A6) was obtained from Cell Signaling Technology, rabbit anti–caspase-1 p10 (C-20) was purchased from Santa Cruz Biotechnology, Inc., and rabbit anti-ASC (AL177) was obtained from Adipogen (all antibodies against the human homologues). HRP-coupled sheep anti–mouse IgG was purchased from GE Healthcare, HRP-coupled goat anti–rabbit IgG was obtained from SouthernBiotech, HRP-coupled goat anti–llama IgG was obtained from Bethyl Laboratories, peroxidase-coupled rat anti-HA (clone 3F10) was obtained from Sigma Aldrich, and HRP-coupled mouse anti-GAPDH (clone 6C5) was purchased from Abcam. Fluorescent secondary antibodies were acquired from Life Technologies.

Schmidt F.I., Lu A., Chen J.W., Ruan J., Tang C., Wu H, & Ploegh H.L. (2016). A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly. The Journal of Experimental Medicine, 213(5), 771-790.

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LPS-Induced Macrophage Polarization

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THP-1-derived M1- and M2-like Mφs were stimulated by 100 ng/ml E.coli K12-LPS (Invivogen, Toulouse, France) for defined time periods at 37^o C/5% CO_2. Afterwhich time, the conditioned supernatants were harvested and stored at -20^o C until required for cytokine assay by sandwich ELISA, whereas cell lysates were used for detection of gene expression by Real Time polymerase chain reaction (RT-PCR) and intracellular proteins by Western blotting.

Al-Shaghdali K., Durante B., Hayward C., Beal J, & Foey A. (2019). Macrophage subsets exhibit distinct E. coli-LPS tolerisable cytokines associated with the negative regulators, IRAK-M and Tollip. PLoS ONE, 14(5), e0214681.

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Evaluating Macrophage Response to Antimicrobial Peptides

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PMA, marimastat, and PMSF were from Sigma Chemical Co. (St. Louis, MO, USA), E. coli K12 LPS, Pam3CSK4, ODN2006, and Quantiblue were from InvivoGen (San Diego, CA, USA). Anti‐phospho‐p38 MAPK, anti‐IκBα, anti‐phospho‐SAPK/JNK, anti‐NF‐κB (p65), anti‐phospho‐Akt (Ser473), and anti‐Akt pan Abs were from Cell Signaling Technology (Danvers, MA, USA). marimastat and Ly294002 (Cell Signaling Technology) solutions were prepared at 1000× in DMSO. Carrier‐free TNF‐α (Cell Signaling Technology) was suspended at 2 μg/ml in PBS containing 5% HI FBS. Synthetic RTD‐1 hydrochloride (>99%) was prepared as described previously [20]. Human α‐defensin HNP‐1 was purified from neutrophils [28]. Stock solutions of RTD‐1, S7 peptide, and HNP‐1 were prepared in sterile 0.01% acetic acid [26, 29].

Tongaonkar P., Trinh K.K., Schaal J.B., Tran D., Gulko P.S., Ouellette A.J, & Selsted M.E. (2015). Rhesus macaque θ‐defensin RTD‐1 inhibits proinflammatory cytokine secretion and gene expression by inhibiting the activation of NF‐κB and MAPK pathways. Journal of Leukocyte Biology, 98(6), 1061-1070.

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Toll-like receptor signaling assay

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PAM3CSK4, FSL-1, Salmonella enterica subsp. enterica serovar Typhimurium flagellin (FLA-ST), poly(I·C), and E. coli K-12 LPS were obtained from InvivoGen. TLR ligands were reconstituted in endotoxin-free H₂O. IFN-α and IFN-β were purchased from PBL InterferonSource. B18R was purchased from Abcam. BAY 11-7082, celastrol, U0126, PD95809, and SB203580 were purchased from Sigma and reconstituted in dimethyl sulfoxide (DMSO). Dynasore was purchased from Tocris Bioscience and was reconstituted in DMSO.

Viglianti G.A., Planelles V, & Hanley T.M. (2021). Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the Long Terminal Repeat. Journal of Virology, 95(7), e02141-20.

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Transwell Assay for Immune Cell Interactions

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The inserts from a 24-well transwell plate (no. 3414, Corning, Inc., Corning, NY) were put into the inserts from a 12-well transwell plate (no. 3401, Corning, Inc., Corning, NY) and both inserts were put into the 12-well transwell plate bottom. 200 µl of DC culture was added to 800 µl LGM-3 and put into the bottom well. 200 µl of GE KER (GE375) were added to 300 µl LGM-3 and put into the middle 12 mm transwell insert. 200 µl of activated HTL were put into the top 6 mm transwell insert. Single cell cultures and multi-two cell cultures were used as controls and were placed in the same well configurations as that in the multi-three cell cultures. Laboratory multi-cell cultures were incubated at 37 °C in 5% CO₂ for 2 hours before adding appropriate agonists.
10.0 µg/ml E. coli K12 LPS (InvivoGen, San Diego, CA) and 10.0 µg/ml Pam3CSK4 (InvivoGen, San Diego, CA), and PBS were added to the middle transwell inserts and plates were incubated at 37 °C in 5% CO₂ for 64 hours.

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Immune Cell Activation Experiments

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Pam2CSK4 and E. coli K12 LPS were purchased from InvivoGen (San Diego, CA, USA). E. coli O111:B4 LPS was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used for flow cytometric analysis or Western blotting were purchased from BioLegend or Cell Signaling Technology (Beverly, MA, USA), respectively. Carboxyfluorescein diacetate succinimidyl ester (CFSE) and Alexa Fluor^® 546 monoclonal antibody labeling kits were obtained from Invitrogen (Grand Island, NY, USA). U0126 and JAK inhibitor were purchased from Calbiochem (La Jolla, CA, USA).

Kang S.S., Kim S.K., Baik J.E., Ko E.B., Ahn K.B., Yun C.H, & Han S.H. (2018). Staphylococcal LTA antagonizes the B cell-mitogenic potential of LPS. Scientific Reports, 8, 1496.

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LFVll Binding to E. coli LPS and Lipid A

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Sixty micrograms of lFVII was mixed with 250 µg of E. coli K12 LPS (Invivogen) or 280 µg of E. coli F583 lipid A (Sigma-Aldrich) in 400 µL of saline, and the resulting mixture was incubated overnight at 37 °C with shaking at 160 rpm. The supernatant of reaction mixture was then collected following 10 min of centrifugation at 17,000 × g before dialysis against distilled water. The purified lFVII and lFVII-untreated E. coli K12 LPS or E. coli F583 lipid A were prepared as controls.

Chen J., Li X., Li L., Zhang T., Zhang Q., Wu F., Wang D., Hu H., Tian C., Liao D., Zhao L., Song D., Zhao Y., Wu C, & Song X. (2019). Coagulation factors VII, IX and X are effective antibacterial proteins against drug-resistant Gram-negative bacteria. Cell Research, 29(9), 711-724.

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