BMDMs were prepared as described previously (63 (link)). Briefly, bone marrow cells were isolated and grown in IMDM media supplemented with 10% FBS in the presence of 20 ng/ml M-CSF (PeproTech) or 5 ng/ml GM-CSF (PeproTech) for 7 days. BMDMs were pretreated with cp-TLR-i for 1 h and activated with 200 ng/ml LPS (Sigma) for the indicated times. For cocultivation with T cells, CD4 T cells were purified from the spleen using anti-CD4 microbeads and MACS column (Miltenyi Biotec). For Th1 polarization, M-CSF-derived BMDMs were treated with 200 ng/ml LPS for 6 h, washed twice with fresh IMDM containing 10% FBS, and cocultured with CD4 T cells in the presence of 2.5 μg/ml anti-CD3ε (BD Biosciences). GM-CSF-driven BMDMs were activated with LPS and cocultured with CD4 T cells in the presence of anti-CD3ε and neutralizing antibodies against IL-12 and IFN-γ (BD Biosciences) to induce Th17 polarization. Four days after the cocultivation, CD4 T cells were restimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 6 h or with 0.5 μg/ml anti-CD3e and 0.5 μg/ml CD28 for 1 day for intracellular cytokine staining and cytokine beads array, respectively.
Lipopolysaccharides (lps)
Manufactured by BD
Sourced in United States
The LPS is a versatile laboratory equipment designed to aid in various scientific applications. It serves as a compact and efficient platform for conducting a range of experiments and analyses. The core function of the LPS is to provide a controlled and consistent environment for sample preparation, processing, and storage, enabling reliable and reproducible results. The specific details and intended use of the LPS may vary depending on the specific requirements of the research or laboratory setting.
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Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (10 mentions)
Lipopolysaccharides (lps), by InvivoGen (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Cytometric bead array, by BD (2 mentions)
Facscalibur, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Cba mouse inflammation kit, by BD (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Flt3l, by R&D Systems (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes buffer, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Lipopolysaccharides (lps), by Promega (1 mentions)
Fugene hd, by Promega (1 mentions)
18 protocols using lipopolysaccharides (lps)
1
Differentiation and Polarization of Bone Marrow-Derived Macrophages
2
Evaluating PAR1 and PAR3 Expression in Whole Blood
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For analysis of PAR1 and PAR3 levels in vivo, whole blood samples of healthy controls were stained with anti-CD 45 (Clone H130, APC/Cy7), anti-CD14 (Clone HCD14, PerCP), anti-CD16 (Clone 3G8, PE, all BioLegend), and the respective anti-PAR1 (clone ATAP2, fluorescein isothiocyanate [FITC]) or anti-PAR3 (clone H-103) antibodies (Santa Cruz). The anti-PAR3 antibody was labeled with a FITC conjugation kit (Abcam). Samples were processed as previously published 16 and measured on a BD Canto II flow cytometer. Note that 100,000 events were recorded and analyzed with the BD Diva 6 software. To investigate if inflammatory activation affects PAR levels on the respective cell surface, whole blood samples were incubated ex vivo with LPS (1 µg/mL, Sigma Aldrich, Missouri, United States) for 4 hours at 37°C and afterwards cells were stained and analyzed as described above. In addition, whole blood samples were incubated with 1 µL/mL GolgiPlug reagent (BD Biosciences, San Jose, California, United States) as recommended by the manufacturer for 30 minutes at 37°C prior to LPS challenge to block transport of newly produced proteins from the Golgi apparatus by brefeldin A. After the stimulation with LPS for 4 hours, blood samples were processed for flow cytometric analysis as described above. Gating was performed as published recently. 16
3
Investigating DC Maturation by Tumor Cells
4
Macrophage Activation by Sporozoite Lysate
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After WT or TLR2−/− mice were intraperitoneally injected with 1 ml 3% TGC (Thioglycollate, Sigma, USA) for three days, peritoneal macrophages were extracted and allowed to adhere on tissue culture dishes for two hours, and non-adherent cells were removed. Next, adherent cells were collected, and 5 × 104 cells were plated in 96-well plates and left at 37 °C overnight. To test the LPS contaminating in SPZ lysate, peritoneal macrophages from the WT mice were then stimulated with 100 ng/ml LPS, 1 × 105 SPZ lysate or equivalent amounts of NSG lysate treated with or without 2.5 μg/ml PmB (polymyxin B, Sigma) for 22 h. To investigate whether SPZ lysate could activate the macrophage in a TLR2-dependent manner, macrophages collected from WT, TLR2−/− and TLR4−/− mice were stimulated with 2 × 105 SPZ lysate or equivalent amounts of NSG lysate or FSL-1(invivogen) or LPS (invivogen) for 17 h. Supernatant was then collected, and the concentration of TNF-a, IL-6 and MCP-1 was determined by FACS flow cytometry using the CBA mouse inflammation kit (BD, USA) according to the manufacturer’s protocol.
5
Isolation and Functional Evaluation of Bone Marrow-Derived Dendritic Cells
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BM cells were isolated from C57BL/6 J female mice by conventional methods35 (link) and suspended at 1.0 × 106 cells/mL in DC culture medium composed of complete RPMI 1640, 10% heat-inactivated FCS (Invitrogen, CA, USA), 2.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 50 μM 2-mercaptoethanol (Invitrogen), 1% non-essential amino acids (NEAA, Invitrogen), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Sigma, MO, USA). The cells were cultured in the presence of 100 ng/mL of Flt-3L (R&D systems, MN, USA) and test extracts, fractions, and compounds were maintained at 37 °C in a 5% CO2 atmosphere. In some assays, BM cells were treated with sample on day 6. On day 7, the cells were stimulated with 5 ng/mL of lipopolysaccharide (LPS) (Sigma). After overnight stimulation, the cells were treated with antibodies, and the expression of cell surface markers was evaluated by flow cytometry using a FACSCanto II instrument BD Biosciences, CA, USA). The production of cytokines IL-12p40 and IL-10 in the supernatant of cell cultures after LPS stimulation was measured by using ELISA kits (BD Pharmingen). In the phase of screening step, each sample was evaluated repeatedly in the two wells, and after identification of 14-DHE, samples at each concentration were evaluated in the three wells.
6
LPS-Induced Cytokine Secretion Assay
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Cells were transduced with virus and differentiated for 14 days followed by stimulation with 100 ng/mL LPS (Sigma, L3024). After 30 min incubation with LPS, 1 μL of Brefeldin A (GolgiPlug, BD Biosciences) was added per 1 mL of culture media containing 1.0E6 cells, according to manufacturer’s instructions. After 6 h of LPS stimulation in the presence of Brefeldin A, the cells were washed with PBS and harvested by treating with 0.5 mM EDTA in PBS for 5–10 min at 37 °C.
7
LPS-Induced Synovial IL-1β Analysis
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BMDCs were cultured in 96-well plates at 1 × 105 cells per well and either treated with 1 μg/ml LPS (InvivoGen), or transfected with 1 μg/ml LPS using 0.3% v/v FuGENE HD (Promega) after priming with 1 μg/ml Pam3CSK4 (InvivoGen) for 6 h. At 16 h after LPS stimulation, the IL-1β concentrations in the culture supernatants were measured using a Cytometric Bead Array (BD Biosciences) in accordance with the manufacturer’s instructions. As for the measurement of IL-1β in the synovium, synovial tissues were resected from the inflamed joints, cut into small pieces, and the tissue weight was measured. The resultant synovial tissues were placed into 500 μl of D-PBS (−) (Nacalai Tesque), then vortexed and centrifuged. The collected supernatants were then used for the measurement of the IL-1β concentration, which was performed using a Cytometric Bead Array, and the results were normalized with tissue weights of individual samples.
8
Pravastatin Regulates Trophoblast Invasion
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The HTR-8/SVneo cells were kindly provided by Dr. Charles Graham from Queen’s University, Canada. The cells were maintained in standard culture conditions at 37 °C in a humidified 5% CO2 incubator. The culture medium (HyClone, South Logan, USA) was RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100μg/mL streptomycin[5 (link)]. HTR-8/SVneo cells were adjusted to 5×105 cells/well in 6-well plates. Cultured in 1% FBS for 12 hours, the cells were treated with 100 ng/mL of LPS (Sigma-Aldrich, St. Louis, USA) to mimic the inflammation induced PE placenta; additionally, 20μg/mL pravastatin[18 (link)] (Sigma-Aldrich, St. Louis, USA) was added as a treatment group. LPS and pravastatin were dissolved in the same RPMI 1640 with 10% FBS.
After 24 hours treatment, the cells were plated in transwell inserts on Matrigel (BD, New Jersey, USA)-coated polycarbonate filters (pore size=8.0μm, Millipore). After 36-hour-incubation, the uninvaded cells were removed with cotton swabs, and the lower surfaces of the filters fixed in 4 g/100 mL paraformaldehyde and stained with 0.2% crystal violet. The data were presented as the number of invaded cells per field viewed using Leica DMR microscope.
After 24 hours treatment, the cells were plated in transwell inserts on Matrigel (BD, New Jersey, USA)-coated polycarbonate filters (pore size=8.0μm, Millipore). After 36-hour-incubation, the uninvaded cells were removed with cotton swabs, and the lower surfaces of the filters fixed in 4 g/100 mL paraformaldehyde and stained with 0.2% crystal violet. The data were presented as the number of invaded cells per field viewed using Leica DMR microscope.
9
Sepsis-Induced Effects on Intestinal Epithelial Cell Migration
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The traditional endotoxicosis model of sepsis was used to determine the effect of sepsis-associated overwhelming innate immune response on intestinal epithelial cell migration in vivo43 (link)46 (link). Briefly, mice were injected intraperitoneally with LPS (2 mg/kg; serotype 0111:B4; Sigma, St Louis, MO) in normal saline. For labeling intestinal epithelial cells in crypts, mice were intraperitoneally injected with BrdU (50 mg/kg, BD Biosciences) at 24 h after LPS treatment. Mice were sacrificed with CO2 inhalation at 48 h after BrdU administration. The entire small intestine was removed, flushed with cold saline, fixed with 10% buffered formalin, and processed for paraffin embedding and sectioning. Sections (5 μm) were processed for BrdU staining and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) using our standard protocol6 (link). Then, BrdU-labeled cells were visualized and analyzed as described before6 (link).
10
Cytokine and Immune Cell Profiling
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For the in vitro experiments, markers of inflammation were measured using the BD CBA Mouse Th1/Th2 Cytokine Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Macrophages, CD4+ T cells, and their subtypes were labeled with anti-mouse antibodies as CD3+, CD4+, CD11b+, CD11c+, CD206+, F4/80+, CD25+, CD44+, CD69+, CD152+, and Foxp3+ (BD Biosciences). Cells were treated with LPS (1 μg/mL, 50 μL; BD Biosciences) to induce differentiation. The FACSCalibur Flow Cytometer (BD Biosciences) and FCAP were used to analyze cells based on the manufacturer’s instructions.
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