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Lipopolysaccharides (lps)

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The LPS is a laboratory instrument designed for the extraction and purification of lipopolysaccharides (LPS) from bacterial samples. It utilizes a combination of chemical and physical techniques to isolate LPS, a key component of the outer membrane of Gram-negative bacteria. The LPS extraction process is a fundamental step in many microbiology and immunology studies.

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Lab products found in correlation

Glutamax supplement, by Thermo Fisher Scientific (1 mentions) Il 4 and il 13, by R&D Systems (1 mentions) Ova peptide, by InvivoGen (1 mentions) Orlistat, by Cayman Chemical (1 mentions) Low molecular weight poly 1 c, by InvivoGen (1 mentions) Lipid a, by Avanti Polar Lipids (1 mentions) Pam3csk4, by InvivoGen (1 mentions) Rmgm csf, by Thermo Fisher Scientific (1 mentions) Interleukin 4 (il 4), by R&D Systems (1 mentions) Cd40l, by R&D Systems (1 mentions) Interleukin 4 (il 4), by RnD Systems (1 mentions) Ifn γ, by BD (1 mentions) Flagellin, by InvivoGen (1 mentions) Peptidoglycan from staphylococcus aureus, by Merck Group (1 mentions) Tlrl picw, by InvivoGen (1 mentions) Anti ly6g microbead kit, by Miltenyi Biotec (1 mentions) Biotinylated rat anti mouse cd144, by BD (1 mentions) Recombinant mouse m csf, by R&D Systems (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions)

9 protocols using lipopolysaccharides (lps)

1

Bone Marrow Derived Macrophage Differentiation and Stimulation

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Bone marrow cells were plated to a density of 5 x 106 cells per 90-mm bacterial Petri dish (Sterilin) in 10ml of DMEM/F-12 Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 with GlutaMAX supplement (Gibco) supplemented with 10% FBS, antibiotics, 20% L-cell conditioned medium, L-Glutamine (1%), HEPES (1%), Sodium Pyruvate (1%) and β-mercaptoethanol (1%). After 4 days of culture, 10ml of additional medium was added and cells were cultured for a further 3 days. Non-adherent cells were washed away and the adherent cells were collected in 5ml PBS with 5% FBS and 2.5mM EDTA. For experiments the cells were re-plated in medium with 1% FBS without L-cell supplement and were incubated overnight before stimulation. Cells were stimulated with IL-4 and IL-13 (20ng/ml) (R&D systems) or LPS (100ng/ml) (Alexis Biochemicals). In some experiments, BMDM were generated from three individual mice and co-cultured with a pool of naïve OTII CD4+CD44−Il4gfp–T cells during stimulation with OVA peptide (323–339) (Invivogen). In some of these co-culture experiments, BMDM were pre-treated for 6 hours with Orlistat (100μM; Cayman), prior to co-culture.
Kannan Y., Perez-Lloret J., Li Y., Entwistle L.J., Khoury H., Papoutsopoulou S., Mahmood R., Mansour N.R., Ching-Cheng Huang S., Pearce E.J., Pedro S. de Carvalho L., Ley S.C, & Wilson M.S. (2016). TPL-2 Regulates Macrophage Lipid Metabolism and M2 Differentiation to Control TH2-Mediated Immunopathology. PLoS Pathogens, 12(8), e1005783.
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2

Preparation and Handling of TLR Ligands

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TLR ligand stocks were prepared as suggested by the manufacturer and stored at −80°C. Working stocks were diluted into complete DMEM at 10X concentration before use. TLR ligand sources: LPS was from Alexis Biochemicals, San Diego, CA, Salmonella minnesota R595 TLRgrade, cat# ALX-581-008-L002; Pam3CSK4 was from InvivoGen, San Diego, CA cat# tlrl-pms; R848 was from InvivoGen, cat# tlrl-r848; poly(I:C) (low molecular weight) was from Invivogen, cat# tlrl-picw; lipid A was from Avanti Polar Lipids, Alabaster, AL, cat# 699500P.
Lin B., Dutta B, & Fraser I.D. (2017). Systematic Investigation of Multi-TLR Sensing Identifies Regulators of Sustained Gene Activation in Macrophages. Cell systems, 5(1), 25-37.e3.
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3

Generating Bone Marrow-Derived Dendritic Cells

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Bone marrow- derived dendritic cells (BMDC) were prepared according to standard protocol. Briefly, mice were sacrificed and tibiae were removed. Marrow from tibiae was isolated and erythrocytes were lysed. Cells were washed and resuspended in DC medium (RPMI 16–40, 10%FBS, 1% Pen/strep, 50uM 2-ME, L-glutamine 2mM) plus 20ng/ml rmGM-CSF (Peprotech) at a concentration of 2x106 cells per 100mm dish. After 10 days, cells were replated at 1x106/ml for experiments in DC medium without GM-CSF. For the relevant experiments cells were stimulated with 10ng/ml LPS (Alexis Biochemicals) or with freshly prepared primary apoptotic cells at a ratio 1:1.
Androulidaki A., Wachsmuth L., Polykratis A, & Pasparakis M. (2018). Differential role of MyD88 and TRIF signaling in myeloid cells in the pathogenesis of autoimmune diabetes. PLoS ONE, 13(3), e0194048.
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4

Splenic B cell stimulation conditions

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Splenic B cells were purified using the EasySep mouse B‐cell isolation kit according to manufacturer instructions. Purity was assessed by flow cytometry, and 90–95% of the purified cells were CD19+. Cells were cultured at appropriate concentrations (i.e., 250,000 cells/well in 200 μl in 96‐well plates) in complete IMDM at 37°C, 7% CO2. Cells were treated with α‐IgM, concentrations between 10 and 1.25 μg/ml (Fab fragment; Jackson Immunores. Lab.), IL‐4 20 ng/ml (R&D Systems), CD40L 20 ng/ml (R&D Systems), LPS 1 μg/ml (Alexis Biochemicals), BAFF 100 ng/ml (Peprotech), CpG 1 μg/ml (ODN1668TypB; Source Bioscience), FICZ 250 nM (Enzo Life Sciences), CH223191 3 μM (Calbiochem), BI605906 10 μM (kindly provided by S. Ley).
Villa M., Gialitakis M., Tolaini M., Ahlfors H., Henderson C.J., Wolf C.R., Brink R, & Stockinger B. (2016). Aryl hydrocarbon receptor is required for optimal B‐cell proliferation. The EMBO Journal, 36(1), 116-128.
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5

Macrophage Activation by Hemozoin

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Macrophages were pretreated with hemozoin and stimulated with - IFNγ [100 ng/ml] (BD Pharmingen), LPS [1 μg/ml] (Alexis Biochemicals) and IL4 [20 ng/ml] (RnD Systems), and processed for Western immunoblotting for iNOS as described previously [19 (link)].
Ranjan R., Karpurapu M., Rani A., Chishti A.H, & Christman J.W. (2016). Hemozoin Regulates iNOS Expression by Modulating the Transcription Factor NF-κB in Macrophages. Biochemistry & molecular biology journal, 2(2), 10.
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6

Immune Cell Culture and TLR Ligand Preparation

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RAW G9 cells were maintained in DMEM, 10% FBS, 20 mM Hepes, and 2 mM glutamine. THP1 B5 cells were maintained in RPMI1640, 10% FBS, and 2 mM glutamine containing 500 ug/ml G418. TLR ligand sources: LPS was from Alexis Biochemicals, Salmonella minnesota R595 TLRgrade, ALX-581-008-L002; Pam3CSK4 (P3C) was from EMC Microcollections, cat# L2000; PGN was from Sigma, peptidoglycan from Staphylococcus aureus, Cat.# 77140; R848 was from InvivoGen, tlrl-r848; Pam2CSK4 (P2C) was from EMC Microcollections, cat# L2020; Flagellin was from Invivogen, FLA-ST ultrapure, tlrl-epstfla; Lipid A was from Avanti Polar Lipids, 699500P; CpG was from IDT; poly I:C was from Invivogen, tlrl-picw.
Li N., Sun J., Benet Z.L., Wang Z., Al-Khodor S., John S.P., Lin B., Sung M.H, & Fraser I.D. (2015). Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages. Scientific Reports, 5, 9559.
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7

Isolation and Infection of Immune Cells

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Primary cells were isolated from wild-type (WT), Fcerg1-/- (encoding FcRγ) [40 (link)] or Mincle-/- [29 (link)] mice on a C57Bl/6J background. Alveolar macrophages (AMΦs) were obtained by bronchoalveolar lavage from mouse lungs. For isolation of alveolar epithelial cells (AECs), lung homogenates were prepared and leukocytes and endothelial cells were depleted from the cell suspension by incubation with biotinylated rat anti-mouse CD45, CD16/32 and CD31 (BD Pharmingen, Heidelberg, Germany) followed by magnetic separation. Microvascular endothelial cells (MVECs) were isolated from lung homogenates by positive magnetic selection using biotinylated rat anti-mouse CD144 (BD Pharmingen). For culturing, plates were coated with fibronectin. Bone marrow-derived macrophages (BMMs) were prepared from the bone marrow and cultured in RPMI 1640 containing 30% L929 cell supernatant and 20% FCS for 10 days. Polymorphonuclear leukocytes (PMNs) were isolated from the bone marrow using the anti-Ly6G MicroBead Kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Cells were infected with S. pneumoniae ST2 (D39) or were stimulated with TDM (Sigma, St. Louis, USA) or LPS (Alexis Biochemicals, San Diego, USA).
Rabes A., Zimmermann S., Reppe K., Lang R., Seeberger P.H., Suttorp N., Witzenrath M., Lepenies B, & Opitz B. (2015). The C-Type Lectin Receptor Mincle Binds to Streptococcus pneumoniae but Plays a Limited Role in the Anti-Pneumococcal Innate Immune Response. PLoS ONE, 10(2), e0117022.
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8

Bone Marrow-Derived Macrophage Activation Assay

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Mice were maintained in specific-pathogen-free conditions and all procedures were performed according to guidelines and study protocols approved by the Institutional Animal Care and Use Committee, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). BMDMs from wild type C57BL/6 mice were prepared by in vitro differentiation for 6 days in complete Dulbecco’s modified Eagle’s medium (DMEM, 10% (v/v) fetal bovine serum (FBS)) at 37°C and 5% CO2 supplemented with 60 ng/ml recombinant mouse M-CSF (R&D). Cells were treated at various time points with 100 nM Dex (Sigma) and/or 10 ng/ml LPS (Alexis Biochemicals, Salmonella minnesota R595 TLRgrade, ALX-581-008-L002). For each assay, cells were preincubated in Hank’s balanced salt solution containing 2% FBS (v/v) at 37°C and 5% CO2 for 2–3 hours before the application of LPS.
Oh K.S., Patel H., Gottschalk R.A., Lee W.S., Baek S., Fraser I.D., Hager G.L, & Sung M.H. (2017). Anti-inflammatory Chromatinscape Suggests Alternative Mechanisms of Glucocorticoid Receptor Action. Immunity, 47(2), 298-309.e5.
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9

Microglia Activation by Amyloid-β and LPS

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Primary murine microglia were stimulated with or without lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO) or human Aβ1–42 (rPeptide, Bogart, GA) prepared in distilled water and used immediately as a mixture of conformations. Cells were preincubated 30 min with or without 1 μM FK506 (Alexis Biochemicals, Enzo Life Sciences, Farmingdale, NY) or vehicle control (DMSO) prior to Aβ or LPS stimulations. BV2 cells were transfected with control, NFATc1, or NFATc2 siRNA using nucleofection. Cells were stimulated with either 10 μM scrambled Aβ1–42 or different concentrations of monomeric Aβ1–42 or fibrillar Aβ1–42 at 48-h post transfection. Media from stimulated cells was collected 24-h post-treatment and TNF-α levels quantified using commercial ELISA (R&D Systems, Minneapolis, MN).
Manocha G.D., Ghatak A., Puig K.L., Kraner S.D., Norris C.M, & Combs C.K. (2017). NFATc2 Modulates Microglial Activation in the AβPP/PS1 Mouse Model of Alzheimer’s Disease. Journal of Alzheimer's disease : JAD, 58(3), 775-787.
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