To activate the polarisation of macrophages, macrophages were stimulated by lipopolysaccharide (LPS).33 Briefly, RAW 264.7 macrophage cells (the National Experimental Cell Resource Sharing platform, Beijing, China) CM was prepared as follows: RAW 264.7 macrophage cells were seeded at 4 × 106 in 10‐cm tissue culture‐treated dishes. They were cultured with DMEM completed medium overnight before being treated with 1 μg/mL lipopolysaccharide (LPS, Escherichia coli serotype 0111: B4, Sigma‐Aldrich) for 24 h. Cell supernatant was discarded and RAW 264.7 macrophage cells were washed three times with phosphate buffer saline (PBS, HyClone, Thermo Scientific). After removing the supernatant, RAW 264.7 macrophage cells were cultured again in DMEM completed medium for another 24 h. The cell culture medium was then collected and centrifuged to remove cell debris. The remaining supernatant was designated as CM and utilized for subsequent experiments.
Lps escherichia coli serotype 0111 b4
Manufactured by Merck Group
Sourced in United States, Germany
LPS (Escherichia coli serotype 0111:B4) is a lab equipment product. It is a bacterial endotoxin derived from the cell wall of Escherichia coli. The core function of this product is to serve as a research tool for the study of inflammatory responses and immune system activation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lta staphylococcus aureus, by Merck Group (2 mentions)
Interferon gamma ifn γ, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Cell culture reagents, by Thermo Fisher Scientific (2 mentions)
Zoletil 50, by Virbac (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Hyclone, by Thermo Fisher Scientific (1 mentions)
Etomoxir, by Merck Group (1 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Anti cd40, by Thermo Fisher Scientific (1 mentions)
6 diazo 5 oxo l norleucine, by Merck Group (1 mentions)
Ku0063794, by Bio-Techne (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
L α aminoadipate l aa, by Merck Group (1 mentions)
Silencer select, by Thermo Fisher Scientific (1 mentions)
Rat elisa kit, by R&D Systems (1 mentions)
Involucrin, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine plus, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
54 protocols using lps escherichia coli serotype 0111 b4
1
Macrophage Polarization Protocol
2
Autophagy Regulation in Bone Homeostasis
3
Neutrophil Recruitment Induced by LPS
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The recruitment of neutrophils to the lungs using LPS was as described previously [39 (link)]. In brief, mice were challenged with aerosolized PBS or 1 mg/mL LPS (Escherichia coli, serotype 0111:B4, Sigma-Aldrich Ltd. Poole, UK) for 30 min. 6 h post-challenge mice were euthanized and BAL collected. The lungs were collected and digested using collagenase as described previously [40 (link)]. Cells were cytospun onto glass slides and differential counts obtained by staining using Wright-Giemsa.
4
Monocyte Cytokine Responses to LPS and LTA
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An amount of 1 × 105 CD14+ cells in RPMI were seeded per well into 96-well cell culture plates and incubated for 24 h at 37 °C in either RPMI media alone, RPMI with 1 µg/mL lipopolysaccharide (LPS; Escherichia coli serotype 0111:B4, Sigma, St. Louis, MO, USA), or 10 µg/mL lipoteichoic acid (LTA; Staphylococcus aureus, Sigma, St. Louis, MO, USA). Culture conditions included 5% CO2. The timing of stimulation was based on prior studies indicating that 24 h stimulation was optimal for monocyte production of innate inflammatory cytokines. After incubation period was over, the plates were centrifuged at 2000 rpm for 10 min after which time supernatant was collected from each well and stored at −80 °C.
5
Mice LPS-Induced Sepsis Model
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Mice were intraperitoneally injected with 15 mg/kg of LPS (Escherichia coli serotype 0111:B4; Sigma–Aldrich) as described elsewhere27 (link),32 (link). After LPS injection, the survival of the mice was monitored. Peritoneal cells and fluids were collected at 0, 3 and 6 hours after LPS injection, as described elsewhere32 (link).
6
Intradermal and Intrathecal Itch Induction
7
Endotoxemia Induction in Mice
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Mice were intraperitoneally (i.p.) injected with 1 mg/kg per body weight (b.w.) lipopolysaccharide (LPS, Escherichia coli, serotype 0111:B4; Sigma‐Aldrich, Saint Louis, MO, USA) in 0.9% saline (NaCl) to induce endotoxemia48 (link). Mice were subsequently subjected to sample collection or intravital imaging as detailed below. Some of their counterparts were untreated (healthy controls, 0 h).
8
HDAC 1-3 Silencing in RAW 264.7 Macrophages
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In order to downregulate the expression of HDACs 1, 2 and 3, cells were subjected to HDAC 1–3 selective siRNAs as follows. One day prior to transfection, RAW 264.7 macrophages were seeded at 20,000 cells/cm2 to obtain identical cell density at the start of the experiment. siRNA transfection experiments were performed in a 12-well plate upon complexing 50 nM siRNA with 3.5 μl Lipofectamine® 2000 (LF2K, Life technologies) according to the manufacturer’s protocol. After 24 h RAW 264.7 macrophages were washed twice with ice-cold PBS and harvested for RNA isolation or Western blotting. Where indicated, cells were stimulated with 10 ng/ml lipopolysaccharide (LPS, Escherichia coli, serotype 0111:B4; Sigma–Aldrich, Zwijndrecht, The Netherlands) and 10 ng/ml interferon gamma (IFNγ, #315-05; PeproTech, Hamburg, Germany) for the last 4 h of the experiment. The following siRNAs were used: mouse HDAC 1 (s119559), HDAC 2 (s67417), HDAC 3 (s67421) and Negative Control siRNA (4390843). All siRNAs used were Silencer® Select Pre-Designed & Validated siRNAs purchased from Ambion® by Life Technologies.
9
Evaluating IL-35-ADSC Therapy for LPS-Induced ALI
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Eighteen mice (8–10 weeks) were anesthetized via isoflurane inhalation in a small sealed box and randomly received 8 × 105 IL-35-ADSCs (in 200-μL sterile normal saline (NS)), 8 × 105 GFP-ADSCs (in 200-μL NS) or isovolumetric NS via tail vein injection (6 mice per group) 7 days before LPS administration. Another 6 mice received nothing and were used as the sham group. The LPS-induced ALI model was constructed in mice in accordance with previous reports (Zhang et al. 2018b (link)). All 18 mice received intratracheal (i.t.) instillation of a single dose of 100-μg LPS (Escherichia coli serotype 0111: B4, Sigma-Aldrich) in 50-μL NS. Mice in the sham group were administered 50-μL NS instead of LPS. The four groups were named Sham, saline + LPS (negative control), GFP-ADSCs + LPS (positive control) and IL-35-ADSCs + LPS, respectively. All mice were anesthetized through intraperitoneal injection of chloral hydrate (330 mg/kg), and bronchoalveolar lavage fluid (BALF) and abdominal aortic blood were collected 24 h post LPS exposure. Then, the mice were killed via cervical dislocation, and lung, spleen and peripheral blood samples were harvested. The weight of the mice was recorded at different time points. The wet/dry weight ratio of the right lung was also calculated.
10
Quantifying Microglia Cytokine Release
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IL-1 β and TNF-α levels were measured in the supernatant of LPS-stimulated or unstimulated microglia cultures in 96-well plates. LPS (Escherichia coli serotype 0111: B4; Sigma-Aldrich) was applied at a concentration of 1 µg/ml. After 6- or 24-h incubation for stimulation of TNF-α or IL-1 β release, respectively (per methods described by Kowalski et al., 2003 (link); Kowalski et al., 2004 (link)), the culture medium was harvested and centrifuged (2,000 × g, 5 min). Cytokine quantification was performed using rat ELISA kits (R&D Systems, USA) following the manufacturer's instructions. Absorbance was measured at 450 nm using the plate reader Mulitskan RC (Labsystems). Intra-assay precision, as measured using coefficient of variation for TNF-α, IL-1β was 7.4% and 8.7%, respectively. Sensitivity of determination for both TNF-α and IL-1β was 5 pg/ml. The data were obtained from three independent experiments (i.e., three independent microglia cultures established from pups of three different dams in each group, for a total of n = 9 per group).
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