HEK293 cells, human hepatoma Huh7 cells, mouse embryonic fibroblasts (MEFs) and murine macrophage cell line RAW264.7 were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) in a 5% CO2 atmosphere at 37℃. HeLa cells were cultured in DMEM (Invitrogen) supplemented with 4% FBS (Thermo Fisher Scientific) in a 5% CO2 atmosphere at 37℃. Primary hepatocytes were prepared as described51 (link). Transfections of HEK293, Huh7 cells or HeLa cells with siRNAs (GenePharma, Shanghai; listed in Supplementary Table S1 ) or plasmids were carried out with lipofectamine 2000 (Invitrogen) or FuGene6 (Roche, in HeLa cells) according to the manufacturer's protocol. The cells were incubated for 72 h to down-regulate p65 or DICER. The knockdown effect was determined by quantitative RT-PCR and Western blot analysis. Ammonium pyrrolidine dithiocarbamate (APDC, 100 ng/ml), BAY 11-7082 (BAY, 30 μM), BMS-345541 (BMS, 1 μM) and LPS (1 μg/ml) were purchased from Sigma, and TNF-α (10 ng/ml) from R&D. All other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co, Ltd (Shanghai, China) unless otherwise specified.
Ammonium pyrrolidine dithiocarbamate
Manufactured by Merck Group
Sourced in United States
Ammonium pyrrolidine dithiocarbamate is a chemical compound used in various laboratory applications. It functions as a chelating agent, capable of forming stable complexes with a range of metal ions.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bms 345541, by Merck Group (1 mentions)
Bay11 7082, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Sirnas, by GenePharma (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
Wortmannin, by MedChemExpress (1 mentions)
Enhanced chemiluminescence ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Fujifilm (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
Origin software, by OriginLab (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Hoechst 33342, by Santa Cruz Biotechnology (1 mentions)
Sodium hydrogen sulfide, by Merck Group (1 mentions)
Ag490, by Merck Group (1 mentions)
Cytochrome c cyt c, by Proteintech (1 mentions)
10 protocols using ammonium pyrrolidine dithiocarbamate
1
Cell Culture and Transfection Protocols
2
Apoptosis Regulation by PI3K/Akt/NF-κB Signaling
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Fetal bovine serum (FBS) and RPMI-1640 medium were obtained from HyClone (GE Healthcare Life Sciences). Acridine orange (AO), Hoechst 33258, ammonium pyrrolidinedithiocarbamate (PDTC) and rhodamine 123 were purchased from Sigma-Aldrich (Merck KGaA). Wortmannin was purchased from MedChem Express. Polyclonal antibodies against PI3K (cat. no. 20584-1-AP), Akt (cat. no. 10176-2-AP), NF-κB p65 (cat. no. 10745-1-AP), IκB (cat. no. 10268-1-AP), GAPDH (cat. no. 10494-1-AP), lamin B (cat. no. 12987-1-AP), caspase-3 (cat. no. 19677-1-AP), poly-(ADP-ribose) polymerase (PARP; cat. no. 13371-1-AP) and inhibitor of caspase-activated DNase (ICAD; cat. no. 10191-2-AP) were obtained from ProteinTech Group, Inc. Phosphorylated (p)-PI3K (cat. no. 4228), p-Akt (cat. no. 4060) and p-NF-κB p65 (cat. no. 3033) antibodies were from Cell Signaling Technology, Inc. Horseradish peroxidase (HRP)-conjugated secondary antibodies (whole IgG affinity-purified antibodies, cat. no. 115-035-003) were obtained from Jackson ImmunoResearch Laboratories, Inc. Enhanced chemiluminescence (ECL) western blotting substrate was from Thermo Fisher Scientific, Inc., and the Annexin V-FITC/propidium iodide (PI) staining kit was from 7 Sea Biotech, Inc.
3
Lipid Synthesis Protocol Compounds
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Palmitic acid (PA) was obtained from Sigma (St. Louis, MO, USA). Glycerol was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Ammonium pyrrolidine dithiocarbamate (PDTC) was obtained from Sigma.
4
Inflammatory Biomarker Quantification in Epithelial-Endothelial Coculture
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The effluent of the medium was analyzed for a panel of intercellular cell adhesion molecule (ICAM-1) and human beta defensin-2 (HBD2) using custom ELISA assay kits (Abbkine, Wuhan, Hubei, CN). Analyte concentrations were determined according to the manufacturer’s instructions using enzyme-labeled instrument coupled with Origin software (OriginLab, Northampton, MA, USA). For inflammation stimulation experiments, healthy epithelia were treated with LPS (10 μg mL−1) or TNF-α (10 ng mL−1) for 24 h, and amounts of secreted ICAM-1 and HBD2 were measured 24 h after treatment. For periodontitis drug studies in chips containing cocultures of human gingival epithelium and endothelium, the endothelial cells were treated with 25 mM NF-κB inhibitor PDTC (Ammonium pyrrolidinedithiocarbamate, Sigma-Aldrich, St. Louis, MO, USA) or blank medium under for 4 h before LPS (10 μg mL−1) or TNF-α (10 ng mL−1) was delivered into the epithelium channel for 24 h. The vascular effluents and epithelium effluents were then collected for ICAM-1 and HBD2 analysis.
5
Extraction and Characterization of S. orientalis L.
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The reagents lipopolysaccharide (LPS, E. coli serotype O55:B5), indomethacin (IND), and ammonium pyrrolidinedithiocarbamate (PDTC), obtained from Sigma-Aldrich (St. Louis, MO, USA), were all of analytical grade and dissolved in phosphate buffer saline as a stock. The S. orientalis L. samples were purchased from a local herbal store (Yuanshan Company, Kaohsiung, Taiwan). The sample's original was identified and its DNA polymorphism had been reported [17 ]. In this study, the samples were freeze-dried and then ground into powder. The dried powder (9.3 kg) was extracted with a 5-fold volume of 95% ethanol by stirring at room temperature for 1 day. This step was repeated three times. The extracted solutions were collected and filtered through filter paper (Whatman number 1; Whatman Paper Ltd., Maidstone, Kent, UK). The SOE was acquired by removing solvent via a rotary evaporator and dried in a freeze dryer. The dry weight of this extract was 489 g, and the yield was 5.3%. The SOE was dissolved in dimethyl sulfoxide (DMSO; the final DMSO concentration never exceeded 0.1% in medium) for cell culture test and was dissolved in sunflower oil for tube feeding in the mice experiment.
6
Inhibition of Oxidative Stress Pathway
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Sodium hydrogen sulfide (NaHS), N-acetyl-cysteine (NAC, an inhibitor of reactive oxygen species, ROS), Ammonium pyrrolidinedithiocarbamate (PDTC, a NF-κB inhibitor), AG 490 (a JNK2 inhibitor) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The anti-cleaved caspase-3 and -9, Bcl-2, cytochrome c (Cyt c), and GAPDH were from proteintech (Wuhan, China). Hoechst 33342 were from Santa Cruz (Bergheimer, Germany). The anti-IκBα-NF-κB and JNK2-STAT3 antibodies were obtained from Cell Signaling Technology (Danvers, USA). Assay kits for malondialdehyde (MDA), superoxide dismutase (SOD), Glutathione peroxidase (GSH-PX), catalase (CAT), lactate dehydrogenase (LDH), creatine kinase (CK) and ROS were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals were from Sigma or Santa Cruz.
7
Investigating NFκB Pathway Modulation
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The cells were allowed to grow for 24h after seeding. Subsequently, they were treated with different concentrations of LDC3/Dynarrestin (diluted in culture medium) for either 1h, 6h, 24h or 48h.
For the activation of NFκB cells were incubated with TNFα (Recombinant Human TNF-α, Peprotech Germany, Hamburg Germany, Cat.# 300-01A) for 6h in a concentration of 100ng/ml. The NFκB inhibition was performed with Ammonium pyrrolidine dithiocarbamate (Sigma-Aldrich, Cat.# P 8765, Munich, Germany) for 6h in a concentration of 50μM. In the case of dual incubation, both compounds were applied at the same time.
The cells were also treated with Sorafenib (LC Laboratories, Woburn, USA, Cat.# S-8599), Dasatinib (LC Laboratories, Woburn, USA, Cat.#. D-3307), 1B Inhibitor (Calbiochem Cat.# 539741) and Ciliobrevin A (Selleckchem, Cat.# S8249).
For the activation of NFκB cells were incubated with TNFα (Recombinant Human TNF-α, Peprotech Germany, Hamburg Germany, Cat.# 300-01A) for 6h in a concentration of 100ng/ml. The NFκB inhibition was performed with Ammonium pyrrolidine dithiocarbamate (Sigma-Aldrich, Cat.# P 8765, Munich, Germany) for 6h in a concentration of 50μM. In the case of dual incubation, both compounds were applied at the same time.
The cells were also treated with Sorafenib (LC Laboratories, Woburn, USA, Cat.# S-8599), Dasatinib (LC Laboratories, Woburn, USA, Cat.#. D-3307), 1B Inhibitor (Calbiochem Cat.# 539741) and Ciliobrevin A (Selleckchem, Cat.# S8249).
8
Antimony Detection Utilizing Carbon Nanofibres
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Analytical grade chemicals and ultrapure water were employed during the experiments. Antimony single standard (1000 mg L−1), ammonium pyrrolidine dithiocarbamate (APDC), iron (II) chloride tetrahydrate, carbon nanofibres (CNFs), hydrochloric acid, iron (III) chloride hexahydrate, ethanol, ammonia (25%), tetraethyl orthosilicate (TEOS), ethanol, 2-methoxy ethanol, antimony(III) chloride, styrene, ethylene glycol dimethacrylate (EGDMA), chloroform, 1,1′-azobisisobutyronitrile (AIBN) and nitric acid (65%) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
9
Multimodal Pharmacological Inhibition
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Doxorubicin (D1515), nifedipine (N7634), autocamtide 2-related inhibitory peptide (AIP, A4308), and ammonium pyrrolidinedithiocarbamate (PTCD, P8765) were purchased from sigma. Amlodipine (A2353) was purchased from Tokyo Chemical Industry. 1,2-Bis (2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid (BAPTA, ab120503) was obtained from abcam.
10
Trace Metal Analysis Methodology
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Organic solvents (i.e., 1-undecanol, chloroform, 1-butyl-3-methylimidazolium hexafluorophosphate, acetone, methanol, absolute ethanol and 1-propanol) and chelating agents (i.e. diethyldithiocarbamate (DDTC), 2-theonyltrifluoroacetone (TTA), ammonium pyrrolidine dithiocarbamate (APDC) and a multi-element 200 mg L -1 organometallic solution were purchased from Sigma-Aldrich (Steinheim, Germany). Sodium chloride, 69% w w -1 nitric acid, 36% w w -1 hydrochloric acid, 85% w w -1 phosphoric acid, sodium dihydrogen phosphate, acetic acid and sodium acetate were obtained from Panreac (Barcelona, Spain). An ICP-IV multi-element 1000 mg L -1 solution was provided by Merck (Darmstadt, Germany).
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