Tlrl eblps

Manufactured by InvivoGen

Sourced in United States

Tlrl-eblps is a research tool for cell culture and molecular biology applications. It functions as a sensor for specific molecular patterns associated with pathogens. The core purpose of this product is to detect and study these patterns in a laboratory setting.

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Lab products found in correlation

Tlrl eklps, by InvivoGen (2 mentions) Imiquimod, by InvivoGen (2 mentions) C5275, by Merck Group (2 mentions) Ifn γ, by InvivoGen (2 mentions) Penicillin, by Fujifilm (1 mentions) Streptomycin, by Fujifilm (1 mentions) Rpmi 1640, by Fujifilm (1 mentions) Fetal bovine serum (fbs), by Nichirei Biosciences (1 mentions) Calyculin a, by Fujifilm (1 mentions) Tak 242, by Cayman Chemical (1 mentions) Fluostar omega, by BMG Labtech (1 mentions) Quanti blue, by InvivoGen (1 mentions) Il 12p40, by Thermo Fisher Scientific (1 mentions) Il 10, by Thermo Fisher Scientific (1 mentions) Il 12p70, by Thermo Fisher Scientific (1 mentions) Spectramax m5 elisa reader, by Molecular Devices (1 mentions) Zymosan a, by Merck Group (1 mentions) Curdlan, by InvivoGen (1 mentions) Tnf α, by Thermo Fisher Scientific (1 mentions) Il 23p19, by Thermo Fisher Scientific (1 mentions)

26 protocols using tlrl eblps

THP-1 monocyte immune response assay

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Briefly, THP-1 human monocytes (TIB-202; ATCC) were cultured in RPMI-1640 (189-02025; Wako) supplemented with 10% fetal bovine serum (175012; Nichirei), penicillin (100 U/mL) (168-23191; Wako), and streptomycin (100 mg/mL) (168-23191; Wako). F. nucleatum was cultured as previously described (43 (link)). THP-1 monocytes were pretreated with 100 μM FAC (RES20400-A7; Sigma-Aldrich), 100 μM DFO (D9533; Sigma-Aldrich), or the indicated concentration of calyculin A (038-14453; Wako) for the indicated time, followed by stimulation with 100 ng/mL LPS (tlrl-eblps; InvivoGen) for the indicated time. THP-1 monocytes were differentiated into macrophages using 6 ng/mL phorbol 12-myristate 13-acetate (AG-CN2-0010; AdipoGen) for 48 hours. Differentiated cells were pretreated with 100 μM FAC, 100 μM DFO, or 5 μM TAK-242 (13871; CAYMAN) for the indicated times, followed by treatment with F. nucleatum at a multiplicity of infection of 10 for 3 hours.

Yamane T., Kanamori Y., Sawayama H., Yano H., Nita A., Ohta Y., Hinokuma H., Maeda A., Iwai A., Matsumoto T., Shimoda M., Niimura M., Usuki S., Yasuda-Yoshihara N., Niwa M., Baba Y., Ishimoto T., Komohara Y., Sawa T., Hirayama T., Baba H, & Moroishi T. (2022). Iron accelerates Fusobacterium nucleatum–induced CCL8 expression in macrophages and is associated with colorectal cancer progression. JCI Insight, 7(21), e156802.

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Assessing NF-κB Activation in THP-1 Macrophages

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Following overnight incubation, THP-1 dual reporter cells were induced to M0 macrophage differentiation with 15 ng/ml PMA for 24 h. Upon differentiation, cells were stimulated for 24 h with 10 ng/ml LPS from different bacterial strains: O. splanchnicus 57; B. fragilis type strain; E. coli K12 (tlrl-eklps InvivoGen; United States), smooth LPS from E. coli 0111:B4 (tlrl-eblps, Invivogen; United States), semi-rought LPS from E. coli R515 and rough LPS from E. coli J5 (IAX-100-007-M001 and 014-M001, respectively, Adipogen; United States). PBS was used as a control. Following 24-h stimulation, the supernatant was collected for the measurement of NF-κB activity.
THP-1 dual cell line NF-κB activity was measured according to the manufacturer’s instructions (InvivoGen; United States). Briefly, the reporter cells have a stable integrator secreted alkaline phosphatase (SEAP) reporter gene for monitoring nuclear factor (NF)-κB activation. Upon NF-κB activation, the SEAP reporter gene is activated, leading to the secretion of alkaline phosphatase, which is then quantifiable by a colorimetric assay (QUANTI-Blue, InvivoGen; United States) using a microplate reader (FluoStar Omega, BMG Labtech; Germany) with absorbance set for 630 nm.

Hiippala K., Barreto G., Burrello C., Diaz-Basabe A., Suutarinen M., Kainulainen V., Bowers J.R., Lemmer D., Engelthaler D.M., Eklund K.K., Facciotti F, & Satokari R. (2020). Novel Odoribacter splanchnicus Strain and Its Outer Membrane Vesicles Exert Immunoregulatory Effects in vitro. Frontiers in Microbiology, 11, 575455.

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Cytokine Profiling of Myeloid Cells

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1×10⁶ BMDMs or BMDCs per ml were left untreated or stimulated with 1 μg/ml LPS-EB (tlrl-eblps, Invivogen), 50 μg/ml Curdlan (Invivogen), 25 μg/ml zymosan A (Sigma), 100 nM CpG (Invivogen), 5 MOI Bacillus Calmette-Guerin (M. bovis-BCG) (45 (link)), or 1 μg/ml Imiquimod (Invivogen) for 16 h. Cytokine concentrations in supernatants were analyzed with the Ready-Set-Go ELISA kits detecting mouse IL-1β, IL-2, IL-6, IL-10, IL-12p40, IL-12p70, IL-23p19, and TNFα (all eBioscience) according to the manufacturer's instructions and measured at 450 nm with a SpectraMax M5 ELISA reader (Molecular Devices).

Vaeth M., Zee I., Concepcion A.R., Maus M., Shaw P., Portal-Celhay C., Zahra A., Kozhaya L., Weidinger C., Philips J., Unutmaz D, & Feske S. (2015). Ca2+ signaling but not store-operated Ca2+ entry (SOCE) is required for the function of macrophages and dendritic cells. Journal of immunology (Baltimore, Md. : 1950), 195(3), 1202-1217.

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Macrophage Infection Dynamics with L. pneumophila

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iBMDM were seeded 12 h before infection into 24 well plates (Corning) at density 2.5 × 10⁵ per well. Macrophages were infected with ΔflaA or ΔdotA_ΔflaA opsonized with anti-L. pneumophila antibody (Meredian life science, B65051G) at an MOI of 0.5 (ΔflaA) or 10 (ΔdotA_ΔflaA) or dosed with heat-killed ΔflaA (Multiplicity of infection, MOI = 0.5) or 1 μg/ml LPS from E. coli O111:B4 (InvivoGen, tlrl-eblps). Cells treated with bacteria or LPS were centrifuged at 1,000 rpm for 5 min at room temperature and incubated at 37°C in 5% CO₂ for various time-points.

Yang C., McDermot D.S., Pasricha S., Bedoui S., Lenz L.L., van Driel I.R, & Hartland E.L. (2019). Interferon γ receptor downregulation facilitates Legionella survival in alveolar macrophages. Journal of leukocyte biology, 107(2), 273-284.

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Immune Signaling Pathway Assays

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Co-culture assays: thymocytes were isolated from untreated C57BL/6 mice and incubated with Nigericin (10 μM, Tocris, 4312) and LPS (1 ng/ml, Invivogen, tlrl-eblps) for 3 h and co-cultured with 1C9s, ANV42.1 or TE-71 cell lines for 20 h before lysis for qPCR. DAMP stimulation: 1C9s were stimulated with ATP (100 μM, Tocris 3312), HMGB1 (1 μg/ml, Abcam, ab78940), IL1a (50 ng/ml,Tocris, 400-ML-005/CF), IL-33 (50 ng/ml, Tocris, 3626-ML-010/CF), or uric acid (50 μg/ml, Sigma, U2625) for 20 h and lysed for qPCR analysis. UTPγS assays: 1C9s or ANV42, cells were stimulated with UTPγS (100 μM, R&D Systems, 3279), or UTPγS plus AR-C 118925XX (20 μM, Tocris, 4890) for 20 h before lysis for qPCR. BzATP triethylammonium salt (100–300 μM, Tocris, 3312), was used for ATP stimulation.

Kinsella S., Evandy C.A., Cooper K., Cardinale A., Iovino L., deRoos P., Hopwo K.S., Smith C.W., Granadier D., Sullivan L.B., Velardi E, & Dudakov J.A. (2023). Damage-induced pyroptosis drives endog thymic regeneration via induction of Foxn1 by purinergic receptor activation. bioRxiv.

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Monocyte Metabolism Modulation Techniques

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Peripheral blood was collected with informed written consent and ethical approval was obtained from Wales Research Ethics Committee 6 (13/WA/0190). Mononuclear cells were obtained by density gradient centrifugation (Histopaque-1077 (10771), Merck) Human CD14+ monocytes were isolated using CD14 microbeads (130-050-201; Miltenyi Biotec). The purity of monocytes was routinely monitored using anti-CD14 Pacific Blue (clone 63D3; 367122; BioLegend) via flow cytometry.
Monocytes (1.0 × 10⁶/mL) were activated with LPS (10 ng/mL; Ultrapure, tlrl-eblps; Invivogen) and cultured in glucose (G7021) or fructose (F3510; 11.1 mM; Merck) containing glucose-free RPMI (11879020; Thermo Fisher) supplemented with 10% dialysed foetal bovine serum (FBS; A3382001; Thermo Fisher Scientific). 2-DG (0.1–1 mM; D8375), oligomycin (1 µM; 75351), antimycin A (1 µM; A8674) and rotenone (1 µM; R8875) were obtained from Merck. The ACLY inhibitor, BMS303141 (4609), was purchased from Tocris. LPS purchased from Invivogen (Escherichia coli K12; tlrl-eklps) was used for the varying glucose concentration experiments.
Cells were harvested and analysed for flow cytometry (acquired with NovoExpress V1.4.1) and supernatants were stored at −20 °C for cytokine analysis.

Jones N., Blagih J., Zani F., Rees A., Hill D.G., Jenkins B.J., Bull C.J., Moreira D., Bantan A.I., Cronin J.G., Avancini D., Jones G.W., Finlay D.K., Vousden K.H., Vincent E.E, & Thornton C.A. (2021). Fructose reprogrammes glutamine-dependent oxidative metabolism to support LPS-induced inflammation. Nature Communications, 12, 1209.

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Pneumonia Induction in Endotoxemic Mice

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Mice were given an i.p. injection of saline as a control or 1 mg/kg of body weight of ultrapure LPS (LPS-EB from E. coli O111:B4; catalog code tlrleblps; InvivoGen, San Diego, CA) to induce endotoxemia. After 18 h, intratracheal (i.t.) instillations of bacteria to induce pneumonia were performed as previously described (17 (link)). In brief, mice were anesthetized with ketamine (50 mg/kg of body weight) and xylazine (5 mg/kg), administered by i.p. injection. A 24-gauge catheter was inserted into the exposed trachea, and a 50-μL bolus of saline containing 10⁶ CFUs of Escherichia coli (serotype O6:K2:H1, ATCC 19138; ATCC, Manassas, VA) was instilled into the left bronchus. Mice were euthanized for tissue collection using an isoflurane overdose after either 18 h i.p. saline, 18 h i.p. LPS, or 18 h LPS followed by 24 h i.t. E. coli. Where indicated, 6 h i.t. of 10⁷ CFUs of Staphylococcus aureus (ATCC 25923) was substituted for E. coli.

Odom C.V., Kim Y., Burgess C.L., Baird L.A., Korkmaz F.T., Na E., Shenoy A.T., Arafa E.I., Lam T.T., Jones M.R., Mizgerd J.P., Traber K.E, & Quinton L.J. (2021). Liver-Dependent Lung Remodeling during Systemic Inflammation Shapes Responses to Secondary Infection. Journal of immunology (Baltimore, Md. : 1950), 207(7), 1891-1902.

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Splenocyte Activation and Cytokine Analysis

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Spleens were collected during tissue collection, trimmed of fat and placed in RPMI medium (R8758, supplemented with 10% fetal bovine serum (F7524, Sigma) and 1% penicillin-streptomycin-glutamine 100x (10378-016, Biosciences)). They were then treated with red blood cell lysis buffer (R7757, Sigma), passed through a 70-μm filter and then resuspended in fresh supplemented RPMI medium. Next, 4 ml of splenocytes were seeded in 6-well plates at a concentration of 16 × 10⁶ cells per well. After a 2.5 h rest period, cells were stimulated with lipopolysaccharide (2 μg ml⁻¹, tlrl-eblps, Invivogen) and concanavalin A (2.5 μg ml⁻¹, C5275, Sigma) for 24 h to induce ceiling antigenicity. Then, supernatant was collected and used for inflammatory cytokine analysis.

Ritz N.L., Draper L.A., Bastiaanssen T.F., Turkington C.J., Peterson V.L., van de Wouw M., Vlckova K., Fülling C., Guzzetta K.E., Burokas A., Harris H., Dalmasso M., Crispie F., Cotter P.D., Shkoporov A.N., Moloney G.M., Dinan T.G., Hill C, & Cryan J.F. (2024). The gut virome is associated with stress-induced changes in behaviour and immune responses in mice. Nature Microbiology, 9(2), 359-376.

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Splenocyte Cytokine Profiling by Luminex

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Splenocytes were resuspended at 2 × 10⁶ cells/mL in complete RPMI. A total of 100 µL of the cell suspension was added to a U-bottom plate. A total of 275 µL of 4X antibody (40 µg/mL) solution was prepared and 50 µL of each antibody was added to appropriate wells for a final concentration of 10 µg/mL, and the cells were incubated for 15 minutes at 37°C. A total of 50 µL of 4x LPS (120 ng/mL, InvivoGen, catalog no. tlrl-eblps) was added at 30 ng/mL final concentration and the cells were incubated for 24 hours. The supernatants were collected, and mediators were analyzed using a Th1/Th2 mouse 20-plex Luminex assay (EPX200-26090-901).

Kauffman K., Manfra D., Nowakowska D., Zafari M., Nguyen P.A., Phennicie R., Vollmann E.H., O'Nuallain B., Basinski S., Komoroski V., Rooney K., Culyba E.K., Wahle J., Ries C., Brehm M., Sazinsky S., Feldman I, & Novobrantseva T.I. (2023). PSGL-1 Blockade Induces Classical Activation of Human Tumor-associated Macrophages. Cancer Research Communications, 3(10), 2182-2194.

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Monocyte-Derived Dendritic Cell Generation

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Monocytes were isolated from PBMCs with the StemCell Technology monocyte negative selection kit (Cat# 19359). Isolated monocytes were stimulated for 24hrs with 0.5μg/mL of LPS (Invivogen, Cat# TLRL-eblps) plus 40ng/mL of IFN-γ (Invivogen, Cat#300–134P) or 0.78μg of empty lipid nanoparticles (eLNP) in 1 mL of GM-CSF and IL-4 supplemented medium. Unstimulated control cells were maintained in GM-CSF and IL-4 supplemented medium for 24hrs. Following stimulation, dendritic cells were harvested and analyzed via flow cytometry and supernatants were collected after stimulation and frozen at −80°C

Connors J., Joyner D., Mege N., Cusimano G., Bell M., Marcy J., Taramangalam B., Lin P., Tam Y., Lin P., Weissman D., Kutzler M., Alameh M.G, & Haddad E. (2022). Lipid nanoparticles (LNP) induce activation and maturation of antigen presenting cells in young and aged individuals. Research Square.

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