Lipopolysaccharide (LPS) from E. coli 055:B5, phorbol 12-myristate 13-acetate (PMA), CXCL8, dibutyryl cyclic AMP (db-cAMP), wortmannin, staurosporine, bovine serum albumin (BSA), and anti-BSA antibody were from Sigma Aldrich (St. Louis, MO, USA); heat-inactivated fetal bovine serum (FBS) was from Gemini-Bioproducts (West Sacramento, CA, USA); misoprostol, LTB4, and PAF were from Cayman Chemical (Ann Arbor, MI, USA); equine recombinant granulocyte-monocyte colony-stimulating factor (GM-CSF) was from Kingfisher Biotech (Saint Paul, MN, USA); and Hank’s balanced salt solution (HBSS) was from Thermo Fisher Scientific (Grand Island, NY, USA).
Lipopolysaccharide lps from e coli 055 b5
Manufactured by Merck Group
Sourced in United States
Lipopolysaccharide (LPS) from E. coli 055:B5 is a well-characterized endotoxin commonly used in research. It is a major component of the outer membrane of Gram-negative bacteria. LPS has the ability to elicit strong immune responses in animals and is frequently used as a tool to study inflammation and immune function.
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Lab products found in correlation
Equine recombinant granulocyte monocyte colony stimulating factor gm csf, by Kingfisher Biotech (2 mentions)
Inf γ, by R&D Systems (2 mentions)
Il 1β, by R&D Systems (2 mentions)
N ethylmaleimide, by Merck Group (2 mentions)
Staurosporine, by Merck Group (1 mentions)
Dibutyryl cyclic amp dbcamp, by Merck Group (1 mentions)
Hank s balanced salt solution hbss, by Thermo Fisher Scientific (1 mentions)
Anti bsa antibody, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Misoprostol, by Cayman Chemical (1 mentions)
Polyvinylidene difluoride pvdf membrane 0.45 μm, by Merck Group (1 mentions)
Ecl kit, by Advansta (1 mentions)
Acetonitrile acn, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus medium, by GE Healthcare (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
9 protocols using lipopolysaccharide lps from e coli 055 b5
1
Neutrophil activation and signaling
2
Immunomodulatory Effects of Gymnema Sylvestre
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Lipopolysaccharide (LPS) from E. coli 055: B5, LC–MS and HPLC-grade solvents and fine chemicals were procured from Sigma-Aldrich, USA. SYBR green mix cDNA synthesis kits were purchased from Takara Bioscience, India. ELISA kits (IL-6, CCL2, INF-γ and IL-1β) were obtained from R&D Systems. Primary antibodies were purchased from Cell Signaling Technology, USA, and the corresponding catalogue numbers are mentioned in supplementary table S2. Bicinchoninic acid reagent (BCA kit) was bought from Thermo Scientific, USA. Polyvinylidene difluoride (PVDF) membrane (0.45 μm) was procured from Millipore, USA. Secondary antibodies were procured from Jackson Laboratory, USA, and an ECL kit was purchased from Advansta, Menlo Park, CA, USA. Deionized water (18 MΩ) was used for the preparation of all solutions. Leaves of G. Sylvestre were collected from Chittoor district, Andhra Pradesh, India. The plant specimen was authenticated by a botany professor from Osmania University, Hyderabad, India. LC–MS CHROMASOLVs-grade and HPLC-grade methanol (MeOH) and acetonitrile (ACN) were procured from Sigma-Aldrich (St Louis, USA). Analytical reagent (AR)-grade ammonium acetate, ammonium formate, formic acid, hydrochloric acid (HCl).
3
Anti-inflammatory Activity Evaluation
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The anti-inflammatory activity was measured following the method described by Villalva et al. (2019) (link). In brief, human THP-1 monocytes (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 media with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (Gibco, Paisley, UK). Cells were seeded in a 24-well plate at 5 × 105 cells/well and differentiated to macrophages by incubation with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Madrid, Spain) for 48 h.
The cytotoxic effect of the extracts was tested on differentiated macrophages, following the MTT assay by Mosmann (1983) (link).
For the anti-inflammatory assay, differentiated cells were incubated with 0.05 μg/mL lipopolysaccharide (LPS) from E. coli 055:B5 (Sigma-Aldrich, Madrid, Spain) in the presence of the samples for 24 h. Then, the supernatants were collected and kept frozen at −80°C. The release of cytokines TNF-α, IL-1β, and IL-6 was measured in the supernatants of differentiated cells using an ELISA kit (BD Biosciences, Aalst, Belgium), according to the manufacturer’s instructions.
The cytotoxic effect of the extracts was tested on differentiated macrophages, following the MTT assay by Mosmann (1983) (link).
For the anti-inflammatory assay, differentiated cells were incubated with 0.05 μg/mL lipopolysaccharide (LPS) from E. coli 055:B5 (Sigma-Aldrich, Madrid, Spain) in the presence of the samples for 24 h. Then, the supernatants were collected and kept frozen at −80°C. The release of cytokines TNF-α, IL-1β, and IL-6 was measured in the supernatants of differentiated cells using an ELISA kit (BD Biosciences, Aalst, Belgium), according to the manufacturer’s instructions.
4
Isolation of Human Monocytes
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The Pan Monocyte Isolation Kit was purchased from Miltenyi Biotec, Bergisch Gladbach, Germany. Lipopolysaccharide (LPS) from E. coli (055:B5), RPMI-1640 medium, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), human AB serum, ethylenediaminetetraacetic acid disodium salt (EDTA), penicillin/streptomycin, and accutase were from Sigma Aldrich (St. Louis, MO). Ficoll-Paque PLUS medium was purchased from GE Healthcare (Uppsala, Sweden), MEM (minimal essential medium) α was from Thermo Fisher Scientific (Waltham, MA), human platelet lysate from PL BioScience (Aachen, Germany), gentamycin from Lonza (Basel, Switzerland), and heparin from Ratiopharm (Ulm, Germany). Dulbecco’s phosphate buffered saline (DPBS) with (+/+) or without (−/−) calcium and magnesium was obtained from Life Technologies (Paisley, UK), and annexin V (Anx5) binding buffer was purchased from BD Biosciences (San Jose, CA).
5
Dehydrozingerone Attenuates Inflammatory Response
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Dehydrozingerone (> 99% purity) bought from TCI chemicals and structure of the compound was shown in Fig. S1A, Lipopolysaccharide (LPS from E. Coli 055: B5) purchased from Sigma Aldrich (USA). Dexamethasone was purchased from Sigma-Aldrich, USA. C-DNA synthesis kit, SYBR green mix purchased from Takara bioscience INDIA. Antibodies GAPDH, JNK P-JNK, c-JUN, p-NF-κB, NF-κB, p38, pP38, Histone-H3, IκB, p-IκB, MPO, Neutrophil Elastase were purchased from Cell signalling technology (USA), ELISA kits (IL-6, CCL2, IL-10, INF-γ, Human-IL-8 and IL-1β were bought from R&D systems. Human IL-6 ELISA kit was procured from Elabsciences (Houston, USA). Secondary Antibodies were procured from Jackson Immuno research private limited (USA). TUNEL kit was procured from Merk Millipore.
6
LPS-Induced Inflammatory Response
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Reagents. Lipopolysaccharide (LPS) from E. coli 055:B5, menadione, N-Ethylmaleimide (NEM) were all purchased from Sigma Aldrich. Recombinant human IL-1 sRII was purchased from R&D systems.
7
Generation of Human Macrophages
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Phosphate-buffered saline (PBS), gentamicin, amphotericin B, macrophage serum free medium (MSF) were obtained from Invitrogen (Lofer, Austria). RPMI-1640, human male AB serum (sterile-filtered), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), fetal bovine serum (FBS), accutase, interleukin-4 (IL-4), interferon-γ (IFN-γ), and lipopolysaccharide (LPS) from E. coli (055:B5, purified by gel filtration) were purchased from Sigma-Aldrich (St Louis, MO, USA). Granulocyte macrophage colony-stimulating factor (GM-CSF) was purchased from Novartis (Basel, Switzerland) and macrophage colony-stimulating factor (M-CSF) from Peprotech, Rocky Hill, NJ.
8
Immunoblotting Analysis of Inflammatory Mediators
9
LPS and Influenza A Virus Exposure
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Lipopolysaccharide (LPS) from E. coli 055:B5 was from Sigma. Influenza A/Puerto Rico/8/34 H1N1 virus (PR8) was grown in the allantoic cavities of 10-day-old embryonated chicken eggs and harvested after 48h at 37°C. Glutathione ethyl ester, biotin amide (BioGEE, Invitrogen) was dissolved in DMSO and then diluted to a final concentration of 200 μM in cell-culture medium. Dexamethasone 21-phosphate disodium salt, N-acetyl-L-cysteine, and N-ethylmaleimide were from Sigma-Aldrich. GSH-C4 was from Pepnome Limited, Hong Kong, China.
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