THC, LPS was purchased from Sigma Aldrich (St. Louis, MO, USA). THC was diluted to the final concentrations (0.5, 1, 2, 5 and 10 μM) as suggested by the instruction. LPS was dissolved in PBS containing DMSO (0.01%) to a final concentration of 100 ng/mL. The MSCs was pre-treated by THC for 24 h, while the microglia was stimulated by LPS for 24 h. AM630 (10 μM) and AM251 (10 μM) (Tocris, Avonmouth, UK) were dissolved in PBS containing DMSO (final concentration: 0.01%). Cells were treated with AM630 or AM251 for 24 hours before examination.
Lipopolysaccharides (lps)
Manufactured by Bio-Techne
Sourced in United Kingdom
LPS is a laboratory product manufactured by Bio-Techne. It is a lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Pg01037, by Bio-Techne (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
L 371 257, by Bio-Techne (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Sc 37007, by Santa Cruz Biotechnology (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Htr 8 svneo, by American Type Culture Collection (1 mentions)
Sl327, by Bio-Techne (1 mentions)
Apoptozole, by Selleck Chemicals (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Huvec line, by American Type Culture Collection (1 mentions)
Myd88, by Santa Cruz Biotechnology (1 mentions)
Lfm a13, by Bio-Techne (1 mentions)
12 protocols using lipopolysaccharides (lps)
1
Cannabinoid Effects on Microglia-Induced Inflammation
2
LPS-induced neuroinflammation model
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LPS (serotype O111:B4, ref. L2630 Sigma, Spain) was dissolved in saline and injected i.p. at 0.5mg/kg. THe dose was chosen according to previous reports to induce neuroinflammation (MacDowell et al., 2013 (link)). OEA (10mg/kg, i.p.; synthesized in our laboratory; Giuffrida et al., 2000 (link)) and PEA (10mg/kg, i.p.; Tocris, Spain) were dissolved in vehicle (5% Tween 80 in saline) and injected 10 minutes before LPS administration. The doses were chosen according to previous studies in rodents reporting antiinflammatory/neuroprotective effects (Plaza-Zabala et al., 2010 (link); Ahmad et al., 2012a (link), 2012b ; Zhou et al., 2012 (link)).
3
Investigating Neuroinflammation via LPS and NKCC1
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P90-110 mice were deeply anesthetized with fentanyl (0.05 mg/kg) and mounted into a stereotaxic frame, then were subjected to either single saline or LPS (lipopolysaccharide from Escherichia coli O26:B6; 200 nl of 5 mg/ml, rate = 200 nl/10 minutes; #L8274, Sigma-Aldrich) injections using a glass micropipette [86 (link)]. The coordinates for the injection were anterior–posterior −2.5 mm, lateral +1.5 mm, and ventral −0.25 mm from the bregma. Bumetanide, a specific inhibitor of NKCC1 in the brain, was coinjected with LPS (50 μM; #3108, Tocris). At 24 hours, mice were transcardially perfused with saline, and approximately 0.5 × 0.5 × 0.5 cm sized tissue pieces from the center of each injected cortical region were cut off and collected for cytokine array and flow cytometric analysis. For tissue sectioning, mice were perfused with saline followed by 4% paraformaldehyde in PBS. For real-time quantitative PCR (qRT-PCR) experiments to assess the effect of NKCC1 deficiency on microglial expression of genes, which contribute to ion homeostasis, membrane potential, cell volume regulation, or inflammation, LPS (5 μg dissolved in ACSF) was administered into the cisterna magna in 5 μl final volume, using a glass capillary. At 24 hours, mice were transcardially perfused with saline followed by CD11b+ magnetic cell sorting.
4
Inducing Systemic Inflammation and Neuroinflammation
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To induce systemic inflammation and a consequent neuroinflammation, 5 mg/kg of LPS (Sigma-Aldrich, St. Louis, MO) were i.p. administered as described before [36 (link)]. Three, 4 or 24 h after LPS injections, mice were sacrificed and the midbrain fraction was extracted as detailed below. In some experiments, mice received i.p. injections of PG01037 (30 mg/kg; Tocris) dissolved in PBS 1 h before LPS administration, a dose that has been proven to exert a therapeutic effect attenuating neurodegeneration in mouse models of Parkinson’s disease [31 (link)].
5
Mouse Microglial Cell Inflammatory Response to Oxytocin
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The mouse microglial cell line MG6 (RIKEN Cell Bank, Tsukuba, Japan) was cultured in Dulbecco’s Modified Eagle’s Medium with high glucose (Sigma-Aldrich, Tokyo, Japan) containing 10% fetal bovine serum, 10 µg/mL insulin, and 100 µM β-mercaptoethanol [19 (link),20 (link),21 (link)]. The effects of OT (Abcam, Cambridge, UK) on inflammatory responses were investigated as follows: cells maintained at 37 °C were pretreated for 30 min with 1 µM OT (diluted in sterilized water) or a vehicle control, followed by stimulation with 100 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich, Tokyo, Japan) or LPS vehicle for 24 h in the continued presence of OT or the OT vehicle. To inhibit OT receptor (OTR) signaling, cells were pre-incubated for 30 min with the OTR antagonist (OTRA) L-371,257 (1 µM; Tocris Bioscience, Bristol, UK) [22 (link)], before adding OT and LPS. To inhibit PTP1B or ATF4 signaling, cells were transfected for 24 h with 15 pmol of PTP1B siRNA (sc-36329; Santa Cruz Biotechnology, Santa Cruz, CA, USA), ATF4 siRNA (sc-35113; Santa Cruz Biotechnology), or control scramble siRNA (sc-37007; Santa Cruz Biotechnology) using a Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA) before treatment with OT and LPS. After washing, cells were harvested and subjected to further analyses.
6
Trophoblast Inflammatory Response Assay
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The human trophoblast cell line HTR8/SVneo, originating from human placental trophoblast cells, was purchased from The American Type Culture Collection (ATCC, USA). HTR8/SVneo cells were cultured on cell plate (3736, Corning, Inc., USA) with RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., USA) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc., USA), 1% sodium pyruvate (Invitrogen; Thermo Fisher Scientific, Inc., USA) and 1% penicillin and streptomycin (Invitrogen; Thermo Fisher Scientific, Inc., USA). The cells were incubated at 37 °C in 5% CO2 and allowed to grow to 80% confluence. Subsequently, they were seeded (3 × 105 cells/well, respectively) into 6-well plates and treated with LPS (100 ng/ml; Sigma-Aldrich; Merck KGaA, USA) for 12 h to induce a trophoblastic inflammatory response. The cells were treated with FPR2 antagonist WRW4 (10 μg/ml; Tocris Bioscience, UK) for 30 min prior to LPS addition [16 (link)]. The samples from the cells and the supernatant were collected for subsequent experiments.
7
PTZ-Induced Seizure Assay in Mice
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PTZ dose used in this study (40 mg/kg in young mice and 50 mg/kg in adult mice, in 0.9% saline, i.p. injection volume was 1 ml/100 g body weight) was determined as a dose at which PTZ induced seizures in 75% of naive mice. In brief, 2- and 11-wk-old mice treated with drugs (saline, LPS [1.0 mg/kg/d; Sigma-Aldrich], SL327 [selective inhibitor MEK1 and MEK2; 32 mg/kg/d; Tocris Bioscience], or LPS plus SL327, i.p. injection for 3 d [P10–12 or P70–72; see details in the figure legends]) were individually placed in a Plexiglas box, and convulsive behaviors were analyzed for 30 min after PTZ injection. The time to onset and the duration of tonic-clonic seizures were measured as previously described (Shen et al., 2012 (link)).
8
Modulation of HSP70 in HUVEC Cells
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The HUVEC line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C, in 5% CO2.
Cells were treated with 0, 200, 500, 1000, and 2000 ng/ml LPS (Sigma, St. Louis, MO, USA) for 24 h or treated with 500 ng/ml LPS for 0, 6, 12, and 24 h; then, the concentration of HSP70 was measured. Further, cells were treated with 0, 6.25, 12.5, and 25 μM TRC051384 (Tocris, Minneapolis, MN, USA), an agonist of HSP70, for 4 h; then, cells were treated with 500 ng/ml LPS for 24 h. Treated cells were used for other experiments. For cotreatment with Apoptozole (Tocris, Minneapolis, MN, USA), an inhibitor of HSP70, and SB203580 (Selleck, Radnor, PA, USA), an inhibitor of p38, cells were treated with 10 μM SB203580 and 10 μM Apoptozole for 20 h.
Cells were treated with 0, 200, 500, 1000, and 2000 ng/ml LPS (Sigma, St. Louis, MO, USA) for 24 h or treated with 500 ng/ml LPS for 0, 6, 12, and 24 h; then, the concentration of HSP70 was measured. Further, cells were treated with 0, 6.25, 12.5, and 25 μM TRC051384 (Tocris, Minneapolis, MN, USA), an agonist of HSP70, for 4 h; then, cells were treated with 500 ng/ml LPS for 24 h. Treated cells were used for other experiments. For cotreatment with Apoptozole (Tocris, Minneapolis, MN, USA), an inhibitor of HSP70, and SB203580 (Selleck, Radnor, PA, USA), an inhibitor of p38, cells were treated with 10 μM SB203580 and 10 μM Apoptozole for 20 h.
9
Studying Tec Kinase Role in Renal Tubular Cells
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Mouse renal tubular epithelial cell line NRK-52E cells was obtained from the Chinese Type Culture Collection (Shanghai Institute of Cell Biology, China) and were inoculated in a sterile culture flask containing calf serum high-glucose DMEM at 37°C in a humidified atmosphere of 5% CO2. When growing together like cobblestones, the cells were seeded on 6-well BioFlex collagen-coated culture plates and grown to 80% confluence. The FBS concentration was reduced to 1% 24 hours prior to the experiments. The cells were stimulated with LPS (Sigma, St. Louis, MO) of different concentrations and duration. To study the role of Tec kinase in renal tubular epithelial cells, different concentrations of LFM-A13, a leflunomide metabolite analogue, were added to the cells 1 h before stimulation with LPS.
The reagents used in this study were purchased from the following sources: LFM-A13 from Tocris Bioscience and Tec siRNA from Genepharma Co., Ltd. (Shanghai, China); and all monoclonal antibodies for NF-κB p-p65, p65, IκBα, and p-IκBα were purchased from Protein Tech Group (Chicago, IL, USA). The antibodies for Tec protein, TLR4, MyD88, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
The reagents used in this study were purchased from the following sources: LFM-A13 from Tocris Bioscience and Tec siRNA from Genepharma Co., Ltd. (Shanghai, China); and all monoclonal antibodies for NF-κB p-p65, p65, IκBα, and p-IκBα were purchased from Protein Tech Group (Chicago, IL, USA). The antibodies for Tec protein, TLR4, MyD88, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
10
Phosphate Modulation of Monocyte Activation
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Monocytes were directly stimulated after isolation in either standard RPMI1640 cell culture medium (high [Pi], 5.6 mM Na2HPO4, Gibco, Lifetechnologies) or customized RPMI1640 containing 1 mM Na2HPO4 (low [Pi]) supplemented with 10% FBS (Gibco, Lifetechnologies). 3 × 105 monocytes were seeded in 96-well plates and 100 ng/ml LPS (Invivogen) was used for monocyte “priming”. Inhibitors were pre-incubated for 30–60 min prior to stimulation.
CaSR (established in our group) and NLRP3-deficient (Invivogen) THP-1 cells were cultured in RPMI1640/10% FBS/1% penicillin and streptomycin (pen/strep) and selection antibiotics as indicated in the data sheet. Assays were performed in 24-well plates. 5 × 105 cells/well were plated for differentiation in 50 ng/ml PMA (Tocris)-containing medium for 2 days before LPS-priming (100 ng/ml) and stimulation.
The following reagents and inhibitors were used for several cell culture experiments. Na2HPO4, BaCl2, fetuin-A from FBS were purchased from Merck, CaCl2, sodium phosphonoformate tribasic hexahydrate, from Sigma, YM254890 from Wako chemicals, Calhex231, NPS2143, Latrunculin A, Cytochalasin D from Tocris, Latrunculin B, PAF C-16, LLOMe from CaymanChemicals, MgSO4 from AppliChem, ATP from Roche, DMSO from Serva, N-fMLP from abcam, and CA-074-Me from Selleck-Chem.
CaSR (established in our group) and NLRP3-deficient (Invivogen) THP-1 cells were cultured in RPMI1640/10% FBS/1% penicillin and streptomycin (pen/strep) and selection antibiotics as indicated in the data sheet. Assays were performed in 24-well plates. 5 × 105 cells/well were plated for differentiation in 50 ng/ml PMA (Tocris)-containing medium for 2 days before LPS-priming (100 ng/ml) and stimulation.
The following reagents and inhibitors were used for several cell culture experiments. Na2HPO4, BaCl2, fetuin-A from FBS were purchased from Merck, CaCl2, sodium phosphonoformate tribasic hexahydrate, from Sigma, YM254890 from Wako chemicals, Calhex231, NPS2143, Latrunculin A, Cytochalasin D from Tocris, Latrunculin B, PAF C-16, LLOMe from CaymanChemicals, MgSO4 from AppliChem, ATP from Roche, DMSO from Serva, N-fMLP from abcam, and CA-074-Me from Selleck-Chem.
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