Escherichia coli k 235

Manufactured by Merck Group

Sourced in France, United States

Escherichia coli K-235 is a laboratory strain of the bacterium Escherichia coli. It is commonly used in research and microbiology applications. The strain is well-characterized and can be used for various experimental purposes.

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6 protocols using escherichia coli k 235

Measuring LPS-induced TNFα in Whole Blood and PBMCs

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LPS-induced TNFα was determined in undiluted whole blood (WB) and peripheral blood mononuclear cells (PBMCs) from matched donors (N=9 healthy human subjects). Blood was collected by venipuncture into heparinized tubes (BD 367874) for WB samples and into CPT tubes (BD, 362753) for PBMCs. PBMCs were isolated according to manufacturer's directions and resuspended at 1 million/mL in RPMI-1640 supplemented with 2% heat-inactivated fetal bovine serum (HyClone). WB and PBMC's were pre-incubated with varying concentrations of pexmetinib for 1 hr, 37°C, 5% CO₂ then stimulated with 100 ng/ml LPS (Escherichia coli K-235, Sigma #L2018) for 16 hours at 37°C, 5% CO₂. TNF-α levels in the cell-free supernatants were quantified by ELISA (R&D systems, DTA00C) as a measure of p-p38 inhibition.

Bachegowda L., Morrone K., Winski S.L., Mantzaris I., Bartenstein M., Ramachandra N., Giricz O., Sukrithan V., Nwankwo G., Shahnaz S., Bhagat T., Bhattacharyya S., Assal A., Shastri A., Gordon-Mitchell S., Pellagatti A., Boultwood J., Schinke C., Yu Y., Guha C., Rizzi J., Garrus J., Brown S., Wollenberg L., Hogeland G., Wright D., Munson M., Rodriguez M., Gross S., Chantry D., Zou Y., Platanias L., Burgess L.E., Pradhan K., Steidl U, & Verma A. (2016). Pexmetinib: a novel dual inhibitor of Tie-2 and p38 MAPK with efficacy in preclinical models of myelodysplastic syndromes and acute myeloid leukemia. Cancer research, 76(16), 4841-4849.

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Time-course response of BV-2 cells to LPS

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BV-2 cells were plated at a density of 10⁵ cells per well in sterile 6-well tissue culture plates and maintained for 24 hrs. Upon media change, individual wells of confluent cells were randomly assigned, in triplicate, as controls with normal culture medium or exposed for 4–24 hrs with LPS (10ng/mL or 1µg/mL; Escherichia coli K-235> 55.10⁴ U/mg, Sigma, France) to determine the time course of response for mRNA levels of TNFα, IL-10, and ATX.

Awada R., Saulnier-Blache J.S., Grès S., Bourdon E., Rondeau P., Parimisetty A., Orihuela R., Harry G.J, & d’Hellencourt C.L. (2014). AUTOTAXIN DOWNREGULATES LPS – INDUCED MICROGLIA ACTIVATION AND PRO-INFLAMMATORY CYTOKINES PRODUCTION. Journal of cellular biochemistry, 115(12), 2123-2132.

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Evaluating VSMC-mediated CD8+ T cell activation

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CD8+ T cells of OT-I mice were isolated from the spleen of male and female mouse, by using the mouse CD8+ T lymphocyte enrichment set (BD Biosciences). VSMC ability to activate T cells was measured in supernatant of 50,000 VMSCs cultured with 500,000 CD8+ T cells of OT-I mice, and stimulated for 24 h with Ovalbumin (Ova) and lipopolysaccharide (LPS) 100 ng/mL from Escherichia coli K-235 (Sigma-Aldrich). DCs (as described above) were used as positive control. As VSMC control groups, VSMCs were cultured without OTI T cells or without Ovalbumin, or OTI T cells were stimulated with Ovalbumin without VSMCs.
To test the impact of cytokine combinations, 100,000 CD8+ T cells or CD4+ T cells isolated from spleens of C57BL/6 mice, were stimulated for 24 h with agonistic antibodies against CD3 (0.1 μg/ml), or with the following cytokines (either single or combined): IL-2 (20 ng/ml), IL-18 (20 ng/ml), IL-12 (20 ng/ml), IL-33 (20 ng/ml), IL-15 (100 ng/ml), and TNFα (20 ng/ml). Culture medium was used as a control. In all experiments, the concentration of IFNγ was measured in supernatant using ELISA assays (BD Biosciences).

Simons K.H., de Vries M.R., Peters H.A., Jukema J.W., Quax P.H, & Arens R. (2019). CD8+ T Cells Protect During Vein Graft Disease Development. Frontiers in Cardiovascular Medicine, 6, 77.

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Intranasal LPS-Induced Lung Inflammation

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Mice were anesthetized intraperitoneally with 90 µg ketamine and 8.1 µg xylazine per gram body mass and received 150 µg LPS intranasally (Escherichia coli K-235, Sigma). After one or two days, 10 × 10⁶ GFP⁺ effector T cells were injected via the tail vein. Two to five days after LPS injection, the lungs of such ‘‘LPS-treated mice’’ contained inflammatory foci with GFP⁺CD8⁺ effector T cell infiltrates.

Mrass P., Oruganti S.R., Fricke G.M., Tafoya J., Byrum J.R., Yang L., Hamilton S.L., Miller M.J., Moses M.E, & Cannon J.L. (2017). ROCK regulates the intermittent mode of interstitial T cell migration in inflamed lungs. Nature Communications, 8, 1010.

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Lipopolysaccharide Detection Protocol

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Sodium dodecyl sulfate (SDS), sodium tetraborate, and sodium hydroxide were obtained from Merck (Darmstadt, Germany). Fluorescein isothiocyanate (FITC), (1-3)-β-D-glucans were purchased from Sigma - Aldrich (St. Louis, MO, USA). Lipopolysaccharides from Escherichia coli 0111:B4 (Lot # 024M4019V), Escherichia coli 055:B5(Lot #025M4040V), Escherichia coli 026:B6 (Lot # 053M4060V), Escherichia coli 0127:B8 (Lot # 103M4051V), Salmonella enterica serotype enteritidis (Lot # 064M4035V), Pseudomonas aeruginosa 10 (Lot # 100M4101V), Salmonella enterica serotype typhimurium (Lot # 093M4088V), Escherichia coli J5 (Rc mutant, rough strains) (Lot # 053M4112V), Salmonella typhosa (Lot # 063M4017V), Escherichia coli F583 (Rd mutant, rough strains) (Lot # 055M4005V), Salmonella enterica serotype minnesota (Lot # 064M4015V), Escherichia coli 0128:B12 (Lot # 063M4014V), Klebsiella pneumoniae (Lot # 111M4038V), Escherichia coli K-235 (Lot # 031M4076V), Salmonella enterica serotype abortusequi (Lot # 104M4064V), and Serratia marcescens (Lot # 013M4078V) were purchased from Sigma Aldrich (St. Louis, MO, USA). Buffers were prepared with ultrapure water obtained from Merck Millipore (Millipore, Bedford, MA, USA). All other reagents were of analytical grade. Experiments were carried out using freshly prepared and filtered solutions.

Fung F.M., Su M., Feng H.T, & Li S.F. (2017). Extraction, separation and characterization of endotoxins in water samples using solid phase extraction and capillary electrophoresis-laser induced fluorescence. Scientific Reports, 7, 10774.

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Cytokine response to LPS in RP105 mice

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Blood was collected from the tail vein from WT and RP105^−/− mice and diluted 1∶25 in RPMI 1640 (Invitrogen) supplemented with non-essential amino acids (PAA Laboratories) and glutamax (Invitrogen). Samples were incubated overnight at 37°C, 5% CO2, in absence or presence of LPS (0–75 ng) from Escherichia coli K-235 (Sigma-Aldrich). Cell-free supernatant was collected and TNFα and IL6 levels were measured by ELISA according to the manufacturer’s protocol (BD Biosciences).

Bastiaansen A.J., Karper J.C., Wezel A., de Boer H.C., Welten S.M., de Jong R.C., Peters E.A., de Vries M.R., van Oeveren-Rietdijk A.M., van Zonneveld A.J., Hamming J.F., Nossent A.Y, & Quax P.H. (2014). TLR4 Accessory Molecule RP105 (CD180) Regulates Monocyte-Driven Arteriogenesis in a Murine Hind Limb Ischemia Model. PLoS ONE, 9(6), e99882.

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