Male C57BL/6 mice (7 weeks of age) were acquired from HyoSung Science Inc. (Daegu, Korea). The mice were kept under controlled conditions in a light/dark cycle of 12 h/12 h, a temperature of 20–24 °C, and humidity of 60–70%. After 1 week of adaptation, the mice were randomly divided into three groups, with 8 mice in each group: the control group, the LPS group, and the LPS+CA group. The mice in the LPS group were given a single intraperitoneal injection of LPS (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 10 mg/kg. The LPS+CA group was intraperitoneally injected with carnosic acid (dissolved in dimethyl sulfoxide (DMSO); Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dose of 40 mg/kg, 1 h after LPS injection. The control group received an equal volume of DMSO. All mice were anesthetized and sacrificed 24 h after LPS injection. Blood and kidney tissues were immediately collected. The doses of carnosic acid and LPS were selected based on previous studies [40 (link),61 (link)].
Lipopolysaccharides (lps)
Manufactured by Santa Cruz Biotechnology
Sourced in United States
LPS is a laboratory reagent produced by Santa Cruz Biotechnology. It functions as a purified lipopolysaccharide extracted from bacterial cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (4 mentions)
Macrophage clone stimulating factor, by R&D Systems (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Il 10, by R&D Systems (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Anti cd14 magnetic bead, by Miltenyi Biotec (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Cd40l, by Enzo Life Sciences (1 mentions)
Flowcellect autophagy lc3 antibody based assay kit, by Merck Group (1 mentions)
Aspirin, by Merck Group (1 mentions)
Nigericin, by Cayman Chemical (1 mentions)
Vildagliptin, by Cayman Chemical (1 mentions)
Saxagliptin, by LGC (1 mentions)
Sitagliptin, by Merck Group (1 mentions)
Human il 1β, by R&D Systems (1 mentions)
Etoposide, by Enzo Life Sciences (1 mentions)
28 protocols using lipopolysaccharides (lps)
1
Carnosic Acid Ameliorates LPS-Induced Kidney Injury
2
Resveratrol Mitigates LPS-Induced Keratinocyte Injury
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The human immortalized keratinocyte HaCaT cell line (CL-0090; Procell, Wuhan, China) was grown in minimum Eagle's medium (MEM; Procell) with 15% fetal bovine serum (Thermo Fisher, Waltham, MA, USA) and 1% penicillin/streptomycin (Thermo Fisher) in 5% CO 2 at 37 °C.
To mimic skin wounds in vitro, HaCaT cells were stimulated with various doses (0, 2, 4, 6, 8 and 10 μg/mL) of LPS (Solarbio, Beijing, China) for 6 h, as previously reported [24] . To analyze the function of RES in LPS-induced HaCaT cell injury, the cells were exposed to various doses (0, 2, 5 and 10 μM) of RES (C 14 H 12 O 3 ; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h and then treated with 6 μg/mL LPS for 6 h.
To mimic skin wounds in vitro, HaCaT cells were stimulated with various doses (0, 2, 4, 6, 8 and 10 μg/mL) of LPS (Solarbio, Beijing, China) for 6 h, as previously reported [24] . To analyze the function of RES in LPS-induced HaCaT cell injury, the cells were exposed to various doses (0, 2, 5 and 10 μM) of RES (C 14 H 12 O 3 ; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h and then treated with 6 μg/mL LPS for 6 h.
3
Isolation and Stimulation of Human Monocytes
4
Quantifying Autophagy in Immune Cells
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PBMCs from healthy donors, activate for T cells with CD3/CD28 beads (Dynabeads Thermo Fisher) (1:1), for B cells with anti-IgM (5 µg/mL, Jackson Immuno Research) and CD40L (100 ng/mL, Enzo Life science) and for monocytes activated with IFNγ (20 ng/mL, Enzo Life science) and/or LPS (10 µg/mL, Santa Cruz) (all 24 hr except for T cells which were stimulated overnight). Autophagy levels were measured after 2 hr treatment with bafilomycin A1 (10 nM BafA1) or vehicle. We adapted the FlowCellect Autophagy LC3 antibody-based assay kit (FCCH100171, Millipore) as follows: In brief, cells were stained with surface markers (as above) and washed with Assay Buffer in 96 well U bottom plates. 0.05% Saponin was added to each well and spun immediately, followed by anti-LC3 (FITC) at 1:20 in Assay Buffer, (30–50 µL/ well) at 4°C for 30 min, and washed once with 150 µL Assay Buffer. Stained cells were fixed with 2% PFA before FACS analysis. Autophagic flux was calculated as LC3-II mean fluorescence intensity of (BafA1-Vehicle)/Vehicle.
5
RvD5 and RvD1 Modulate LPS-Induced Inflammation
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RvD5 (7S,17S-dihydroxy-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid), Cayman Chemical, Ann Arbor, MI, USA, 0,1; 1 and 10 ng/animal) or vehicle (0.1% ethanol in saline) were administrated intraperitoneally (i.p.). After 60 min, mice received LPS (10 mg/kg, from E. coli—Santa Cruz Biotechnology) or vehicle (0.9% saline solution) intravenously (i.v.; caudal vein). Animals were divided into five equal sized groups consisting of six animals per group. Group 1: 0.1% ethanol in saline (i.p.) + saline (i.v.); Group 2: 0.1% ethanol in saline (i.p.) + LPS 10 mg/kg (diluted in saline, i.v.); Group 3: RvD5 0.1 ng/animal (diluted saline—i.p.) + LPS 10 mg/kg (i.v.); Group 4: RvD1 1 ng/animal (i.p.) + LPS 10 mg/kg (i.v.); and Group 5: RvD1 10 ng/animal (i.p.) + LPS 10 mg/kg (i.v.). For all assays, samples were collected 6 h after LPS administration.
6
Aspirin Pretreatment in Severe Acute Pancreatitis
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Mice were injected with 7 doses of Cae (50 μg/kg, Ana Spec, Inc., San Jose, CA, USA) at 1-hour intervals intraperitoneally; then intraperitoneal injection with LPS (7.5 mg/kg, Santa Cruz, CA, USA) was carried out one hour later, to induce SAP model.
Animals were randomly assigned to 4 groups (each group had 8–12 mice): control, SAP, L-ASA, and H-ASA. The mice in control group mice were given saline (0.9% NaCl) solution intraperitoneally instead of Cae and LPS. Aspirin (Sigma-Aldrich, St. Louis, MO) suspension liquids were prepared in 0.5% of carboxyl methyl cellulose sodium (CMC-Na, Kemiou Chemical Reagent Corp, Tianjin, China). Two doses of Aspirin (12.5 mg/kg, 125 mg/kg) were used to pretreat SAP, based on average human body weight of 60 kg, and these doses correspond to approximately 100 mg and 1000 mg per day in human, respectively [15 (link), 16 (link)]. Vehicle (CMC-Na) or Aspirin was administered 1 h before the first Cae injection.
Animals were randomly assigned to 4 groups (each group had 8–12 mice): control, SAP, L-ASA, and H-ASA. The mice in control group mice were given saline (0.9% NaCl) solution intraperitoneally instead of Cae and LPS. Aspirin (Sigma-Aldrich, St. Louis, MO) suspension liquids were prepared in 0.5% of carboxyl methyl cellulose sodium (CMC-Na, Kemiou Chemical Reagent Corp, Tianjin, China). Two doses of Aspirin (12.5 mg/kg, 125 mg/kg) were used to pretreat SAP, based on average human body weight of 60 kg, and these doses correspond to approximately 100 mg and 1000 mg per day in human, respectively [15 (link), 16 (link)]. Vehicle (CMC-Na) or Aspirin was administered 1 h before the first Cae injection.
7
Blastocystis sp. ST3 Induced Caco-2 Cell Response
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For this purpose, 1 × 105 Caco-2 cells were counted and seeded in each well of a six-well plate. The plate was incubated in 5% CO2 at 37 °C overnight. After 70–80% confluency, the B3STA prepared from 105 of Blastocystis sp. ST3 was added to the sample well. In addition, 20 ng/mL LPS (Santa Cruz Biotechnology Cat No. sc-3535) were used to compare the induction pattern to the B3STA. A well full of Caco-2 cell without any treatment either by B3STA or LPS, was considered as control group. All groups were in duplicate and evaluated 24 h after exposure.
8
Inducing Endotoxemia Sepsis in Mice
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9
Caspase Activation and Pyroptosis Assays
10
Macrophage Differentiation and Activation
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Bone marrow cells from tibiae and femora of donor mice were incubated for 7 days at 37°C in complete IMDM medium supplemented with 10 ng/ml of macrophage clone-stimulating factor (R&D systems, Abingdon, UK) to obtain adherent macrophages. Briefly, to obtain bone marrow (BM)-derived macrophages, hind legs of 2 mice were cleaned thoroughly, and bone marrow from the femurs and tibiae was flushed using ice-cold PBS. The collected bone marrow cells were pooled, washed once in ice-cold PBS, and resuspended in complete IMDM medium supplemented with 10 ng/ml of macrophage clone-stimulating factor (R&D systems, Abingdon, UK) and cultured for 8 days to differentiate macrophages. Macrophage was then induced by incubating adherent cells for 20 h at 37°C in complete IMDM medium supplemented with 1 ng/ml LPS (Santa Cruz Biotechnology, Inc., CA) in the presence or absence of curcumin (10 μM) according to previous reports [15 (link)–17 (link)]. Then, macrophages were cultured for 30, 60, and 90 min under the previously specified culture conditions. Macrophages were harvested at each specified time point. Total, cytoplasmic, and nuclear proteins of macrophages were isolated and analyzed as detailed above. ELISA was used to measure the level of inflammatory factors.
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