Blood samples were drawn via the PE-50 catheter using sterile technique. In the first stage, 10 μl of blood sample was on 5% sheep blood agar and incubated at 37°C for 24 hours under aerobic conditions to determine the bacterial load. Colony-forming units were quantified by manual counting. Approximately 0.6 ml of blood sample was collected for biochemical assays in two stages of the study. After centrifugation (1000 ×g, 15 minutes, 4°C), the serum was removed and stored at −80°C for further analysis. Serum PCT, HMGB1 (Cusabio Biotech Co. Ltd., Wuhan, China), and cTnI (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were detected using specific enzyme-linked immunoassay kits according to the manufacturer's instructions. Serum BUN and ALT were measured with a Hitachi 7180 biochemistry automatic analyzer (Hitachi, Co. Ltd., Tokyo, Japan).
Hmgb1
Manufactured by Cusabio
Sourced in China
HMGB1 is a protein that functions as a DNA-binding protein. It is involved in the regulation of transcription and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Cusabio (2 mentions)
7180 biochemistry automatic analyzer, by Hitachi (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Myd88, by Proteintech (1 mentions)
Hmgb1, by Proteintech (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Proteintech (1 mentions)
P ikbα, by Affinity Biosciences (1 mentions)
β tubulin, by Proteintech (1 mentions)
Il 1β elisa kit, by Cusabio (1 mentions)
Traf6, by Affinity Biosciences (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
β actin, by Proteintech (1 mentions)
Cck 8 reagent, by Beyotime (1 mentions)
Dihydroethidium dhe, by Beyotime (1 mentions)
Sod assay kit, by Nanjing Jiancheng (1 mentions)
Polybrene, by Hanbio Biotechnology (1 mentions)
Lipid peroxidation malondialdehyde mda assay kit, by Beyotime (1 mentions)
Tgf β, by Cusabio (1 mentions)
7 protocols using hmgb1
1
Quantitative Analysis of Bacterial Load and Serum Biomarkers
2
Inflammatory Protein Expression Quantification
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Mouse p-AMPK (Thr 172) (Catalogue No. MBS7230575, My BioSource, USA), NF-κB (P65) (Catalogue No. MBS775083, My BioSource, USA), high mobility group box protein-1 (HMGB1, catalogue No. CSB-E08224r, CUSABIO Technology LLC, China,), NLRP3 (Catalogue No. LS-F39627, LSBio, USA), caspase-1 (Catalogue No. NBP2-75014, Novus Biologicals, USA) and IL-1β (Catalogue No. MLB00C, R&D systems, USA) protein expression were determined and quantified using ELISA kits according to the manufacture’s protocol.
3
Effects of E. ulmoides Polysaccharide on Oxidative Stress
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E. ulmoides polysaccharide (EUP, content: 60%, batch number TR20180607, extracted from E. ulmoides leaves) was obtained from Xi'an Tianrui Bio-Tech Co., Ltd. (Xi'an, China). The lipid peroxidation malondialdehyde (MDA) assay kit, dihydroethidium (DHE), and CCK-8 reagent were acquired from Beyotime (Shanghai, China), and the superoxide dismutase (SOD) assay kit was acquired from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). Primary antibodies against Toll-like receptor 4 (TLR4), high-mobility group protein B1 (HMGB1), myeloid differentiation factor 88 (MyD88), NF-κB p65, IKB-α, interferon regulatory factor 1 (IRF-1), β-tubulin, β-actin, and horseradish peroxidase- (HRP-) conjugated secondary antibody were purchased from Proteintech (Wuhan, China). Primary antibodies against tumor necrosis factor-receptor-associated factor 6 (TRAF6), P-p65, and P-IKB-α were purchased from Affinity Biosciences (Cincinnati, OH, USA). HMGB1, TNF-α, and IL-1β ELISA kits were obtained from CUSABIO (Wuhan, China). Fetal bovine serum (FBS), RPMI-1640 medium, penicillin, and streptomycin were purchased from Gibco (Rockville, MD, USA). TLR-4 overexpression plasmid, empty plasmid, and polybrene were purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China).
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E. ulmoides polysaccharide (EUP, content: 60%, batch number TR20180607, extracted from E. ulmoides leaves) was obtained from Xi'an Tianrui Bio-Tech Co., Ltd. (Xi'an, China). The lipid peroxidation malondialdehyde (MDA) assay kit, dihydroethidium (DHE), and CCK-8 reagent were acquired from Beyotime (Shanghai, China), and the superoxide dismutase (SOD) assay kit was acquired from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). Primary antibodies against Toll-like receptor 4 (TLR4), high-mobility group protein B1 (HMGB1), myeloid differentiation factor 88 (MyD88), NF-κB p65, IKB-α, interferon regulatory factor 1 (IRF-1), β-tubulin, β-actin, and horseradish peroxidase- (HRP-) conjugated secondary antibody were purchased from Proteintech (Wuhan, China). Primary antibodies against tumor necrosis factor-receptor-associated factor 6 (TRAF6), P-p65, and P-IKB-α were purchased from Affinity Biosciences (Cincinnati, OH, USA). HMGB1, TNF-α, and IL-1β ELISA kits were obtained from CUSABIO (Wuhan, China). Fetal bovine serum (FBS), RPMI-1640 medium, penicillin, and streptomycin were purchased from Gibco (Rockville, MD, USA). TLR-4 overexpression plasmid, empty plasmid, and polybrene were purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China).
4
Quantitative ELISA Analysis of Inflammatory Biomarkers
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With MDA (#A003-1, Nanjing jiancheng Bioengineering Institute), SOD (#A001-3, Nanjing jiancheng Bioengineering Institute), TNF-α (#CSB-E04741m, CUSABIO), IL-1β (#CSB-E08054m, CUSABIO), HMGB1 (#CSB-E08225m, CUSABIO), IL-6 (#CSB-E04639m, CUSABIO), TGF-β (#CSB-E04726m, CUSABIO), and PGE2 (#CSB-E07965h, CUSABIO) quantitative ELISA kits, related factors were tested according to instructions. The DYNATECHMR7000 microplate reader was used to calculate the concentration of each indicator by forming the standard curve through the constant value provided.
5
Measuring Inflammatory Markers in BAL Fluid
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Tumor necrosis factor (TNF)-α (BioLegend, CA), Pro-MMP9 (R&D Systems, Abingdon, UK), interleukin(IL)-6 (BioLegend), IL-1β (BioLegend), S100A8/9 (R&D Systems), and HMGB1 (CUSABIO, Houston, TX) levels in bronchoalveolar lavage (BAL) fluid and Der p-specific immunoglobulin E (IgE) level in serum were measured using enzyme-linked immunosorbent assay according to the manufacturer’s instructions. The concentrations of total, oxidized, and reduced glutathione in serum were calculated using a glutathione detection kit (Enzo Life Sciences).
6
Biomarker Profiling in Sepsis Model
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Approximately 0.1 ml of blood was collected from the tail vein at a defined time after CLP for every biochemical assay in this study. After centrifugation (3000 rpm, 15 min, 4 °C), the serum was removed and stored at −80 °C for analysis. Serum PCT, CRP, cardiac troponin I (cTnI) (USCN Life Science, Inc., Wuhan, China) and HMGB1 (Cusabio Biotech Co., Ltd., Wuhan, China) levels were determined using specific enzyme-linked immunoassay (ELISA) kits following the manufacturer’s instructions. The concentrations of interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) were quantified using ELISA kits (Arigo Biolaboratories, Hsinchu City, Taiwan, China). The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (Cre) were used as indicators for liver and kidney function and were measured with a biochemistry automatic analyzer (Chemray 240, Rayto Life and Analytical Sciences Co., Ltd., China). Serum lactate was detected using the Lactate Assay Kit (Arigo Biolaboratories, Hsinchu City, Taiwan, China).
7
Evaluating Apoptosis and Immune Response
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DOX and GEM were obtained from MedChemExpress (NJ, USA). PLGA was bought from Sigma-Aldrich (MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kyushu, Japan). The Annexin V-FITC Apoptosis Detection Kit was bought from BD Biosciences (NJ, USA). Monoclonal antibodies to CD8, Gr-1, CD11b and calreticulin (CRT) were bought from BioLegend (CA, USA). ELISA kits for transforming growth factor-β (TGF-β), interleukin-10 (IL-10) and high mobility group protein B1 (HMGB1) were bought from CUSABIO (Wuhan, China).
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