α-Hederin of purity over 99% was provided by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). NF-κB specific inhibitor pyrrolidine dithiocarbamate (PDTC), JAK2/STAT3 signaling specific inhibitor AG490, and ERK specific inhibitor U0126 were obtained from Selleck Chemicals (Houston, TX, USA). Dimethyl sulfoxide (DMSO) was used to dissolve the above compounds for experiments, and single treatment with DMSO was used as negative control. Recombinant human IL-6 cytokine was obtained from Solarbio Life Science (Beijing, China). The primary antibodies used for Western blot analyses against cyclin B1, CDK1, Bcl-2, Bax, Cyt c, cleaved-caspase-9, cleaved-caspase-3, cleaved-caspase-8, cleaved-PARP, NF-κB(p65), p-IκBα, IκBα, p-IKKα, IKKα, p-ERK, ERK, COX IV, lamin B1, and GAPDH were purchased from Bioworld Technology, Inc. (MN, USA).
Pyrrolidine dithiocarbamate
Manufactured by Selleck Chemicals
Sourced in United States
Pyrrolidine dithiocarbamate is a chemical compound used in various laboratory applications. It serves as a chelating agent, capable of forming stable complexes with metal ions. The core function of this product is to facilitate the extraction, separation, and analysis of metal species in different sample matrices.
Automatically generated - may contain errors
Lab products found in correlation
U0126, by Selleck Chemicals (1 mentions)
α hederin, by Yuanye Bio-Technology (1 mentions)
Ag490, by Selleck Chemicals (1 mentions)
Cyclin b1, by Bioworld Technology (1 mentions)
Bcl 2, by Bioworld Technology (1 mentions)
Bcl2 associated x (bax), by Bioworld Technology (1 mentions)
Cleaved caspase 9, by Bioworld Technology (1 mentions)
P iκbα, by Bioworld Technology (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Cleaved caspase 3, by Bioworld Technology (1 mentions)
Nf κb p65, by Bioworld Technology (1 mentions)
A740003, by Merck Group (1 mentions)
Ges 1, by Merck Group (1 mentions)
Ges 1, by American Type Culture Collection (1 mentions)
Anti p perk, by Cell Signaling Technology (1 mentions)
Anti chop, by Cell Signaling Technology (1 mentions)
Sp600125, by Cell Signaling Technology (1 mentions)
Anti grp78 bip, by Cell Signaling Technology (1 mentions)
Anti atf4, by GeneTex (1 mentions)
Anti ire1, by Cell Signaling Technology (1 mentions)
5 protocols using pyrrolidine dithiocarbamate
1
α-Hederin Inhibits IL-6-Induced Signaling
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α-Hederin of purity over 99% was provided by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). NF-κB specific inhibitor pyrrolidine dithiocarbamate (PDTC), JAK2/STAT3 signaling specific inhibitor AG490, and ERK specific inhibitor U0126 were obtained from Selleck Chemicals (Houston, TX, USA). Dimethyl sulfoxide (DMSO) was used to dissolve the above compounds for experiments, and single treatment with DMSO was used as negative control. Recombinant human IL-6 cytokine was obtained from Solarbio Life Science (Beijing, China). The primary antibodies used for Western blot analyses against cyclin B1, CDK1, Bcl-2, Bax, Cyt c, cleaved-caspase-9, cleaved-caspase-3, cleaved-caspase-8, cleaved-PARP, NF-κB(p65), p-IκBα, IκBα, p-IKKα, IKKα, p-ERK, ERK, COX IV, lamin B1, and GAPDH were purchased from Bioworld Technology, Inc. (MN, USA).
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α-Hederin of purity over 99% was provided by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). NF-κB specific inhibitor pyrrolidine dithiocarbamate (PDTC), JAK2/STAT3 signaling specific inhibitor AG490, and ERK specific inhibitor U0126 were obtained from Selleck Chemicals (Houston, TX, USA). Dimethyl sulfoxide (DMSO) was used to dissolve the above compounds for experiments, and single treatment with DMSO was used as negative control. Recombinant human IL-6 cytokine was obtained from Solarbio Life Science (Beijing, China). The primary antibodies used for Western blot analyses against cyclin B1, CDK1, Bcl-2, Bax, Cyt c, cleaved-caspase-9, cleaved-caspase-3, cleaved-caspase-8, cleaved-PARP, NF-κB(p65), p-IκBα, IκBα, p-IKKα, IKKα, p-ERK, ERK, COX IV, lamin B1, and GAPDH were purchased from Bioworld Technology, Inc. (MN, USA).
2
Macrophage Activation and NLRP3 Signaling
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Human macrophage cell line U937 (Cell Bank, Chinese Academy of Sciences) was cultured with 5% CO2 for 24 h at 37°C in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (Invitrogen). Phorbol myristate acetate (PMA) (100 ng/ml) was added to stimulate U937 into the adherent macrophage phenotype (PMA-U937). Then, cells were stimulated with 1 mM ATP for 30 min, followed by treatment with 10 μg/ml of T. spiralis ES antigens for 24 h. To evaluate the effect of P2X7R blockade on NLRP3 signals, 10 μM A740003 (antagonist of P2X7R; Sigma) was added before stimulation with ATP. Cells incubated with cell culture medium were used as the control. To determine the role of NF-κB in the activation of NLRP3 by P2X7R, an NF-κB inhibitor pyrrolidine dithiocarbamate (50 μM; Selleck Chemicals, USA) was added before stimulation with ATP. Supernatants and cultured cells were collected for further use.
3
Modeling Gastric Inflammation and Cancer In Vitro
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Normal human gastric epithelial cells (GES-1) and gastric cancer cells (AGS) were acquired from the American Type Culture Collection (Manassas, VA, USA). GES-1 cells were cultured in Dulbecco’s Modified Eagle Medium (Bioagrio, Mountain View, CA, USA) supplemented with 10% fetal bovine serum (Bioagrio) and AGS cells in RPMI 1640 Medium (Bioagrio) with 10% fetal bovine serum.
GES-1 cells at 50% to 70% confluence were serum starved for 24 hours before being exposed to various concentrations of CDCA (Sigma-Aldrich, Darmstadt, Germany) for an additional 24 hours in order to produce CDCA-induced GIM. For inhibition of NF-κB signaling, CDCA treatment was combined with the NF-κB inhibitor pyrrolidine dithiocarbamate (Selleck, Houston, TX, USA; 50 µM) in GES-1 cells. GES-1 cells were given 24 hours of treatment with 15 µg/µL of betulinic acid (MCE, Shanghai, China) to activate NF-κB signaling.
GES-1 cells at 50% to 70% confluence were serum starved for 24 hours before being exposed to various concentrations of CDCA (Sigma-Aldrich, Darmstadt, Germany) for an additional 24 hours in order to produce CDCA-induced GIM. For inhibition of NF-κB signaling, CDCA treatment was combined with the NF-κB inhibitor pyrrolidine dithiocarbamate (Selleck, Houston, TX, USA; 50 µM) in GES-1 cells. GES-1 cells were given 24 hours of treatment with 15 µg/µL of betulinic acid (MCE, Shanghai, China) to activate NF-κB signaling.
4
Investigating the Unfolded Protein Response
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Primary antibodies included anti-RCN1 (Bethyl, Montgomery, TX, USA), anti-GFP (MBL, Nagoya, Japan), anti-Flag (Sigma), anti-IP3R1 (Bethyl), anti-tubulin (Sigma), anti-ATF4 (GeneTex, San Antonio, TX, USA), anti-pIRE1 (Abcam, Cambridge, MA, USA), anti-pPERK, anti-caspase-3, anti-PERK, anti-IRE1, anti-GAPDH, anti-CHOP, anti-eIF2α and anti-GRP78/BiP (Cell Signaling Technology, Danvers, MA, USA). The drugs used were TG (Sigma), TM (Sigma), sp600125 (Cell Signaling), 2-APB (Sigma), Xec, pyrrolidine dithiocarbamate, BAY 11-7082, GSK2606414 and STF883010 (Selleckchem, Houston, TX, USA).
5
Cell Viability Assay Protocol Using MTT
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Cell viability was measured by the MTT assay. Briefly, the cells were plated in a 96-well plate (4 × 10 3 cells/ well). After 24 hr, the cells were treated with DMSO or different concentrations of LPS (Guangzhou Hewei Chemical Co., LTD, Guangdong, China), IKK-16 as a selective inhibitor of IκB kinase, which is more sensitive to IKKβ than IKKα (Selleck Chemicals, Shanghai, China) and pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor kappa B (NF-kappa B) activation (Selleck Chemicals). After different time points of treatment, 100 μL of 5 mg/mL MTT (Sigma-Aldrich, St. Louis, MO, USA) was added to each well for 4 hr, the medium was replaced with 200 μL of Dimethyl Sulphoxide (DMSO), and the cells were incubated at room temperature in the dark for 6 hr. The optical density (OD) value was measured using a spectrophotometric microtiter plate reader at 570 nm. The effect was expressed as percentage relative to the controls.
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