TPPU was provided by Dr. Bruce Hammock (University of California, Davis, CA). Dimethyl sulfoxide (DMSO), Evans blue (EB) and fluorescein isothiocyante (FITC)-dextran were all obtained from Sigma Co. (Sigma-Aldrich, St Louise, MA, USA). Human brain microvascular endothelial cells (HBMVECs) were purchased from Angio-Proteomie (Boston, MA, USA). MTT was purchased from Beyotime Biotechnology (Nanjing, China). Phosphate buffer saline (PBS) and fetal bovine serum (FBS) were obtained from PAN-Biotech (Aidenbach, Germany). 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Pyrrolidinedithiocarbamate (PDTC, a selective NF-kB inhibitor) was obtained from Selleck Chemicals (LLC, Houston, TX, United States).
Pyrrolidine dithiocarbamate pdtc
Manufactured by Selleck Chemicals
Sourced in United States
Pyrrolidine dithiocarbamate (PDTC) is a chemical compound with the molecular formula (C₅H₁₀NS₂). It is a dithiocarbamate derivative that functions as a chelating agent, capable of forming complexes with various metal ions.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Beyotime (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Beyotime (1 mentions)
β actin, by Merck Group (1 mentions)
P c jun, by Abcam (1 mentions)
P c fos, by Abcam (1 mentions)
Sr11302, by Selleck Chemicals (1 mentions)
Enhanced chemiluminescence ecl plus kit, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Sb202190, by Selleck Chemicals (1 mentions)
C fos, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
P iκbα, by Abcam (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Incucyte s3 live cell analysis system, by Sartorius (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
4 hydroxytamoxifen 4oh tam, by Merck Group (1 mentions)
Antibodies against hmgb1, by Abcam (1 mentions)
7 protocols using pyrrolidine dithiocarbamate pdtc
1
Cerebrovascular Endothelial Cell Assays
2
Primary Astrocyte Culture Protocols and Reagents
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The 2-Chloroethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Ningbo, China). Reagents for the primary culture of astrocytes were purchased from Biological Industries (Beit-Haemek, Israel). The quantitative real-time (RT)-PCR assay kit was purchased from Takara, Japan. The enhanced chemiluminescence (ECL) plus kit, bicinchoninic acid (BCA) protein assay kit and NE-PER™ nuclear and cytoplasmic extraction reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). SB202190, pyrrolidine dithiocarbamate (PDTC), and SR11302 were purchased from Selleck (Houston, TX, USA) and APExBIO (Houston, TX, USA). Primary antibodies against MMP-9, p65, IκBα, c-Jun, c-Fos, p-c-Jun, p-c-Fos, p-IκBα, and LaminB were products of Abcam (Cambridge, UK) and Cell Signaling Technology (Beverly, MA, USA). Antibodies against glial fibrillary acid protein (GFAP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-actin were obtained from Millipore (Billerica, MA, USA), Proteintech (Wuhan, China), and ABclonal (Wuhan, China), respectively. The secondary antibodies conjugated with Alexa Fluor 488 or tetramethylrhodamine (TRITC), RIPA Lysis Buffer, and DAPI were obtained from Beyotime Biotechnology (Shanghai, China).
3
BMSC-DC2.4 Co-culture and NF-κB Inhibition
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Mouse primary bone mesenchymal stem cells (BMSCs) and DC2.4 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Hyclone, USA), containing 10% fetal bovine serum (FBS; Hyclone, USA) and 100 U·mL−1 Pen&Strep (Gibco, USA) at 37 °C under humid conditions in a 5% CO2 atmosphere. Pyrrolidine dithiocarbamate (PDTC,10 μmol·L−1, Selleck, USA) was used to block the NF-κB signaling pathway.
4
Quantifying Cellular ROS Levels
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The cellular ROS level was detected using the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), which can be oxidized to a highly fluorescent 2′,7′-dichlorofluorescein (DCF). AGS cells grown to confluence on 60 mm culture dish were treated with H. pylori (MOI 100 wt, cagA, virB7), chemotherapeutics (5 μM CPT, 1 μM DOX, 1 μM STS) or cytokines (10 ng/ml IL-1β, 10 ng/ml TNF) for 4 h. For the analysis of intracellular ROS levels, cells were stained with 20 μM H2DCF-DA for 30 min at 37 °C according to the manufacturer’s protocol and DCF fluorescence was measured by flow cytometry (CyFlow space). Hydrogen-peroxide (H2O2)-treated cells served as positive control and tert-butyl hydrogen peroxide (TBHP) (50 µM) was used as a positive control for analysis setting. Where indicated, AGS cells were treated with 20 or 40 μM of ROS inhibitor pyrrolidine dithiocarbamate (PDTC) (Selleckchem). Data were reported as the mean ± standard deviation of at least n = 3 different experiments. For DCF imaging, images were captured with a 20 × objective in green channel by the IncuCyte® S3 Live Cell Analysis System (Sartorius, Essen Biosciences).
5
Tamoxifen Resistance in Breast Cancer Cells
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Antibodies against HMGB1 were purchased from Abcam. Antibodies against TLR4 and NF‐κB (RelA) were purchased from Boster. CDK4/6 inhibit or abemaciclib was purchased from Selleck. HMGB1 human recombinant protein (for cell culture) was purchased from Boster. 4‐Hydroxytamoxifen (4‐OH Tam) was purchased from Sigma‐Aldrich. HMGB1 inhibitor glycyrrhizic acid (GA) and NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC) were purchased from Selleck. MCF‐7 and T47D cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences and maintained in Dulbecco's modified Eagle's minimal essential medium (DMEM, 4500 mg/L glucose, Gibco) with 10% heat‐inactivated fetal bovine serum (FBS, Sciencell). Tamoxifen‐resistant MCF‐7 (MCF‐7R) cells were obtained by exposing MCF‐7 cells to 4‐OH Tam whose concentration was increased up to 1000 nmol/L for 6 mo. Tamoxifen‐resistant T47D (T47DR) cells were obtained by exposing T47D cells to 4‐OH Tam whose concentration was increased up to 1000 nmol/L for 12 mo. MCF‐7R and T47DR cells were maintained in DMEM with 5% FBS plus 1000 nmol/L 4‐OH Tam.
6
Alveolar Type II Epithelial Cell Analysis
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Human lung alveolar type II epithelial A549 cells were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). LPS (Sigma-Aldrich; Merck KGaA), anti-TGF-β1 antibody (cat. no. ab64715, Abcam), ALK5 inhibitor (TP0427736; cat. no. S8700, Selleck Chemicals), pyrrolidine dithiocarbamate (PDTC; cat. no. S363302, Selleck Chemicals) and immunohistochemistry reagents and kits (Beyotime, China) were used in this study. Other reagents and chemicals were obtained from Western Biotechnology (China).
7
Bone Marrow Stem Cell Culture and NF-κB Inhibition
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Mice bone marrow stem cells were cultured in a-Minimal Essential Medium (MEM) (Hyclone) supplemented with 10% FBS and 100 U/mL penicillin G and 100 mg/mL streptomycin (Hyclone) at 37 °C under humid conditions with 5% CO2. In addition, the NF-κB pathway was blocked by pyrrolidine dithiocarbamate (PDTC) (Selleck, Houston, TX, U.S.A).
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