Ciglitazone (>99.4% purity) was purchased from Tocris Biosciences (Bristol, UK) and GW 9662 (>98% purity) was purchased from Enzo Life Sciences (Farmingdale, NY USA). For both chemicals, stock solutions were prepared in high-performance liquid chromatography (HPLC)-grade dimethyl sulfoxide (DMSO) and stored in 2-mL amber glass vials with polytetrafluoroethylene-lined caps. Working solutions were prepared by spiking stock solutions into sterile cell culture media immediately prior to each experiment, resulting in 0.1% DMSO within all treatment groups.
Ciglitazone
Manufactured by Bio-Techne
Sourced in United Kingdom
Ciglitazone is a chemical compound used as a laboratory reagent. It functions as a selective agonist for the peroxisome proliferator-activated receptor gamma (PPARγ). Ciglitazone is commonly utilized in cell-based assays and research applications involving the study of PPARγ activity and signaling pathways.
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Lab products found in correlation
T0070907, by Bio-Techne (2 mentions)
Gw9662, by Enzo Life Sciences (1 mentions)
Propylene glycol, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Thermo Fisher Scientific (1 mentions)
Bovine thrombin, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sq22536, by Bio-Techne (1 mentions)
Alexa 647 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
15d pgj2, by Enzo Life Sciences (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Gw9662, by Merck Group (1 mentions)
Camp elisa kit, by Enzo Life Sciences (1 mentions)
Sebacic acid, by Merck Group (1 mentions)
Decanoic acid, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Gsk3787, by Bio-Techne (1 mentions)
High glucose dmem culture medium, by Merck Group (1 mentions)
Gw6471, by Bio-Techne (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
5 protocols using ciglitazone
1
Ciglitazone and GW 9662 Stock Preparation
2
Preparation and Use of Chemical Reagents
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Ciglitazone (>99.4% purity) was purchased from Tocris Bioscience (Bristol, UK), and dorsomorphin (DMP) (99.7% purity) was purchased from Millipore Sigma (St. Louis, MO, USA). For both chemicals, stock solutions were prepared in high performance liquid chromatography-grade dimethyl sulfoxide (DMSO) and stored in two mL amber glass vials with polytetrafluoroethylene-lined caps. Working solutions were prepared by spiking stock solutions into particulate-free water from our recirculating system (pH and conductivity of ~7.2 and ~950 μS, respectively) immediately prior to each experiment, resulting in 0.2% DMSO within all vehicle control and treatment groups. Propylene glycol (>99.5% purity) and Oil Red O (ORO) (>75% dye content) were purchased from Fisher Scientific (Hampton, NH, USA) and Sigma–Aldrich (St. Louis, MO, USA), respectively.
3
Platelet Activation and Signaling Pathways
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Fibrinogen, bovine thrombin, H89, GW9662 and IMBX were purchased from Sigma Aldrich (Poole, UK). 15dPGJ2 was purchased from Enzo Life Sciences and Ciglitazone and SQ22536 from Tocris Bioscience (Bristol, UK). The cAMP ELISA kit was from Enzo Life Sciences (Exeter, UK). Primary anti‐ FAK (focal adhesion kinase) (C20), Syk (N‐19), PLCγ2 (Q20), β3 (C20) and actin (C11) antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Phospho‐specific primary antibodies for β3 Y779 and Akt S473 were from Abcam (Cambridge, UK). Anti‐phospho–PKC (protein kinase C) substrate, phospho‐S157 and S239 VASP and phospho‐Ser 19 myosin light chain antibodies were purchased from New England BioLabs (Cell Signalling, Hitchin, UK), and anti‐phospho‐Tyr 4G10 antibody was purchased from Millipore (Watford, UK). Fluorophore conjugated secondary antibodies, Fluo‐4 calcium indicator dye and Alexa‐488 and Alexa‐647 conjugated phalloidin were purchased from Life Technologies (Paisely, UK). All other reagents were from previously described sources 26 .
4
Influence of PPARγ on Histone Modifications
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To determine the influenceability of the histone modifications H3K4me3 and H3K9ac by PPARy, 50,000 HVT respectively primarily isolated EVT cells were seeded in 500 µL medium (RPMI-1640 + 10% FCS) per chamber of a chamber slide. After growing and adhesion to the slides, the cells were treated with the PPARγ-agonist Ciglitazone (20 mM, Tocris Bioscience, Bristol, UK) [69 (link),70 (link),76 (link)] and the PPARγ antagonist T0070907 (50 mM, Tocris Bioscience, Bristol, UK) or respective control vehicle. After an incubation period of 24 h, immunofluorescence staining was performed. The concentrations of the PPARγ-agonist Ciglitazone and the PPARγ antagonist T0070907, as well as the incubation period were chosen according to the literature published with these chemicals [69 (link),70 (link),71 (link),76 (link),77 (link)].
5
Huh7 Cell Culture and Treatments
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Huh7 (Japanese Collection of Research Bioresources Cell Bank, no. JCRB0403, Japan) cells were cultured in DMEM high glucose culture medium (Sigma‐Aldrich Co. LLC. no. D5796) supplemented with 10% FBS (Invitrogen), 1× penicillin/streptomycin (Sigma) and non‐essential amino acids (Sigma) in Normoxic conditions (37°C, 5% CO2). Cells were passaged at 70–80% confluency using 0.05% Trypsin in PBS (Severn Biotech). Cells were used experimentally up to passage 10. For treatment, cells were seeded into 6‐well plates at 2 × 105 cells per well and allowed to recover for 48 h. Treatment compounds were added directly into culture medium. Cells were treated for 4 h either under standard conditions or in stress conditions (2% O2, 10% CO2, 32°C; combined hypoxia, hypercapnia and hypothermia) with a vehicle control (DMSO unless otherwise indicated) or compound of interest: 2‐ene‐VPA (2VPA; MolPort), 2‐propyloctanoic acid (2POA; Sigma), ciglitazone (Tocris), decanoic acid (Sigma), GSK3787 (Tocris), GW6471 (Tocris), octanoic acid (Sigma), sebacic acid (SA; Sigma), T0070907 (Tocris), VPA (Sigma, vehicle dH2O), valpromide (VPD; Katwijk Chemie, The Netherlands).
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