MDA-MB-231 cells were seeded into 96-well plates at 6,000 cells per well and cultured overnight in DMEM + 0.1% FBS. Cells in triplicate wells were then treated with 100 ng/ml rHMGB1 (R&D Systems) in the presence or absence of 10 mg/ml HMGB1-neutralizing antibody (Novus) and then 5 μM Dox or vehicle. Cell viability was measured 24 h later by trypan blue exclusion.
Rhmgb1
Manufactured by R&D Systems
Sourced in United States
RHMGB1 is a recombinant human protein that functions as a growth factor. It is produced in a baculovirus expression system.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by BioLegend (1 mentions)
Lps rs, by InvivoGen (1 mentions)
Anti hmgb1, by BioLegend (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Fps zm1, by Merck Group (1 mentions)
Elisa kit, by Arbor Assays (1 mentions)
Atractylenolide 1, by Merck Group (1 mentions)
Fluoroblok membrane inserts, by BD (1 mentions)
Green cmfda dye, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Srage, by R&D Systems (1 mentions)
Blocker casein, by Thermo Fisher Scientific (1 mentions)
Hmgb1, by R&D Systems (1 mentions)
Phospho mlkl, by Cell Signaling Technology (1 mentions)
Phospho rip, by Cell Signaling Technology (1 mentions)
Phospho rip3, by Cell Signaling Technology (1 mentions)
Phospho rip3, by Abcam (1 mentions)
Phospho mlkl, by Abcam (1 mentions)
Goat anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
14 protocols using rhmgb1
1
Doxorubicin Sensitivity in HMGB1-Treated Cells
2
LPS-Induced Acute Lung Injury Model
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Male mice were anesthetized by intraperitoneal (i.p.) injection of 1% pentobarbital sodium solution (80 mg/kg, Biyuntian Institute of Biotechnology) and randomly assigned to the following groups: Control, LPS, positive control, and treatment group (n=4-6 per each group). Mice in the LPS and treatment groups then received an i.p., injection of LPS (15 mg/kg; Sigma-Aldrich, Merck KGaA) diluted in 200 µl sterile PBS. The control and positive control groups were administered only PBS by the same way and volume. The positive control and treatment groups received an intranasal (i.n.) injection of anti-HMGB1 (2.5 µg/g in 40 µl PBS, 2 h after LPS challenge; BioLegend, Inc.) (22 (link)) or rHMGB1 (0.5 µg/g in 40 µl PBS, 2 h after LPS challenge; R&D Systems, Inc.) (23 (link)-25 (link)), an i.p., injection of LPS from Rhodobacter sphaeroides (LPS-RS), a TLR2/4 antagonist (0.1 mg/mg in 200 µl endotoxin-free water, 1 h before LPS challenge; InvivoGen) (25 (link)-27 (link)) or FPS-ZM1, a RAGE antagonist (1.5 mg/kg in 200 µl PBS, 1 h before LPS challenge; EMD Millipore) (25 (link),28 (link)). All mice were sacrificed (cervical dislocation) 24 h after the last treatment and the blood, lung, bronchoalveolar lavage fluid (BALF), and spleen of mice were extracted for sample preparation.
3
Astrocyte HMGB1 Stimulation and TLR Modulation
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Primary astrocytes were stimulated with 500 ng/mL of rHMGB1 (Cat# 1690-HMB; R&D) in the presence or absence of 10 nM atractylenolide I (TLR4 antagonist; Cat# A2737; Sigma) or 10 nM C29 (TLR2 inhibitor; Cat# 363600-92-4, MCE, Monmouth Junction, NJ, USA) dissolved in 0.1% dimethyl sulfoxide for 24 hours. The culture supernatants were then collected, and the cells were subjected to lysis with a buffer (Beyotime). The lysates were centrifuged at 10,000 × g for 15 minutes. The PGE2 concentration in cell supernatants and lysates was measured using a PGE2 high-sensitivity enzyme-linked immunosorbent assay (ELISA) kit (Arbor Assays, Ann Arbor, MI, USA) according to the manufacturer’s instructions.
4
HMGB1 Modulates Corneal Inflammation
5
Breast Cancer Cell Migration Assay
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The breast cancer cells were fluorescently labeled by incubation with 5 μmol/L Green CMFDA Dye (Invitrogen) for 2 h. Fluoroblok™ membrane inserts (8 μM pore size, BD Biosciences) were coated with 100 μl of 10% Matrigel™ (BD Biosciences) in cold serum-free DMEM. Companion plates with coated inserts were incubated at 37 °C for 2 h. Then, the remaining liquid (coating buffer) from the permeable support membranes was carefully removed without disturbing the layer of Matrigel™ (BD Biosciences) on the membranes. After 2 h, cells were counted and 5 × 104/well in 1% BSA DMEM without or with 100 ng/ml rHMGB1 (R&D Systems) or in a combination with either 150 nM GDC-0941 or 2 μM AT13148 was added into the upper wells of coated 8-μm pore Fluoroblok™ membrane inserts in 24-well companion plates (BD Biosciences). The lower chamber was filled with 800 μl medium containing 5% FBS DMEM. The assay plates were incubated in a humidified atmosphere at 37 °C in a 5% CO2 incubator. Imaging was performed as described in the chemotaxis assay.
6
HMGB1-Mediated Dendritic Cell Activation
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BMDCs were isolated and cultured in DC medium as described previously49 (link). After 8 days of culture, DCs were enriched using anti-CD11c-coated magnetic microbeads (Miltenyi-Biotec, Auburn, CA, USA) and treated with medium in the absence or presence of rHMGB1 (500 ng/ml) (R&D Systems, Minneapolis, MN, USA) with or without various concentrations of sRAGE (10, 100, 200, or 400 ng/ml) (R&D Systems) for 2 days. Cytokine concentrations in the cell culture supernatants were measured using an IL-23 ELISA kit (R&D Systems). rHMGB1 and sRAGE were tested by Limulus amebocyte lysate (ZhanJiang A&C Biological, China) and considered to be endotoxin free. In addition, rHMGB1 was in the disulphide form in this study.
7
Cortical HMGB1 Injection in Rats
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Rats were anesthetized with 60 mg/kg ketamine and 7.5 mg/kg xylazine, and their heads immobilized in a stereotactic frame. Recombinant HMGB1 (rHMGB1) (R&D Systems, Minneapolis, MN), 2.5 μg in 5 μl 1X phosphate buffered saline (PBS) or 5 μl 1X PBS injection was then injected into the overlying cortex using a Hamilton syringe. This dose is in line with HMGB1 doses used in previous HMGB1 injection studies [15 (link)]. Three rats each were used for rHMGB1 or PBS injections. Coordinates for cortical injections were at a depth of 2.5 mm from the overlying cortex, 5.5 mm from the midline, and at −4.52 mm bregma. Rats were euthanized 48 hrs following cortical rHMGB1 injection and transcardiac perfusion performed with 4% paraformaldehyde. Brains were placed in 30% sucrose for cryoprotection. Once brains sank to the bottom of the container, 10 μm cryosections were prepared and sections prepared for immunohistochemistry.
8
Purification and Characterization of Complement Proteins
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Purified sC1s, C1 complex, and C1-INH were purchased from Calbiochem (Billerica, MA, USA). RC1s and rHMGB1, both produced in mouse myeloma cells, were obtained from R&D systems (Minneapolis, MN, USA). His-HMGB1 produced in bacteria was obtained from GenScript, Co. (Piscataway, NJ, USA). LPS (Escherichia coli, serotype 055:B5) and mouse anti-NPM1 (clone FC82291) and anti-β-actin monoclonal antibodies (clone AC-74) were purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA). A rabbit anti-HMGB1 antibody was obtained from Upstate Biotech (Billerica, MA, USA). Rabbit anti-C1s and goat anti-C1q antibodies were obtained from Quidel, Co. (San Diego, CA, USA). Rabbit antibodies for HSP90α and RPLP0 were obtained from Abcam plc (Cambridge, UK).
9
Serum Anti-HMGB1 Antibody Quantification
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Referencing other studies9 (link), the procedure of serum anti-HMGB1 antibody test was as follows: Maxisorp polystyrene 96-wells plates were coated with 50 μl per well of rHMGB1 (R&D Systems, Minneapolis, USA) at 1 μg/ml in PBS and incubated overnight at 4 °C. After one wash, plates were blocked with Blocker Casein (Thermo, Rockford, USA) for 1 h. Serum samples, diluted 1:50 with the sample buffer, were added in duplicate (100 μl/well) and incubated for 2 h at room temperature. After five washes, 100 μl HRP-conjugated rabbit anti-human IgG (Euroimmun, Lubeck, Germany) was added to each well and incubated for 30 min at room temperature. After washing, bound antibodies were detected using 3,3′,5,5′-tetramethylbenzidine dihydrochloride/hydrogen peroxide (TMB/H2O2). The reaction was stopped with 0.5 M sulphuric acid and the absorbance was measured at 450 nm using a microplate-spectrophotometer (Thermo MK3, Rockford, USA). Anti-HMGB1 antibody levels were expressed in relative units.
10
Evaluating HMGB1's Impact on NF-κB Activation
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To assess for the effect of HMGB1 on NF-κB activation by MA, a verification experiment using rHMGB1 (R&D Systems, MN, USA) was conducted. After pretreated with 20 μM MA, the H9c2 cells were treated with recombinant HMGB1 (rHMGB 1) protein (200 ng/ml) or vehicle for 2 h and then, subjected to H/R procedure. The application concentration of rHMGB1 was determined according to the previously published articles (26 (link), 27 (link)). After various treatments, the supernatants and cells were collected to detect the concentration of inflammatory factors by ELISA and the level of apoptotic-related proteins by western blot.
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