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7 protocols using Fasudil

Three hESC lines and four hiPSC lines were maintained, based on the hPSC research guidelines of the Stem Cell Center, Asan Medical Center (Seoul, Republic of Korea). The hPSCs were cultured on vitronectin-coated (5 μg/ml; Life Technologies, Grand Island, NY, USA) culture plates in Essential 8 medium (Life Technologies) at 37°C, 5% CO2. The cultures were passaged every week in clusters by chemically dissociating the PSCs using lab-made reagent (0.4% sodium citrate, S4641, Sigma-Aldrich, St. Louis, MO, USA) or commercial reagent (Gentle Cell Dissociation Reagent, Stem Cell Technologies, Vancouver, BC, Canada). Dissociation using lab-made reagent was carried out as described previously [23 (link)], and for comparison with lab-made reagent, the commercial reagent was used according to the manufacturer’s instructions. The hPSC clusters were transferred onto prepared vitronectin-coated culture plates supplemented with fasudil (10 μM; Adooq, Irvine, CA, USA) or Y-27632 (10 μM; Sigma-Aldrich).
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Stem cells were differentiated by the previously published conventional hepatogenic differentiation protocol [9 ] and an advanced protocol. Briefly, in the advanced protocol, the cells were seeded on 0.1% gelatin-coated dishes at 7000 cells/cm2 in a cell culture medium. After 2 days, they were cultured for 7 days with Step-1 medium consisting of Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco) supplemented with 0.1% polyvinyl alcohol (PVA; Sigma Aldrich) or 1% FBS, 10 mM nicotinamide (Sigma Aldrich), 20 ng/ml hHGF (Peprotech), 10 ng/ml FGF2, 2 μM 5-azacytidine (Sigma Aldrich), 0.1 μM dexamethasone (Sigma Aldrich), 1% insulin-transferrin-selenium (ITS; Gibco), 3 μM CHIR99021, 20 ng/ml EGF (Peprotech), and 10 μM Fasudil (AdooQ Bioscience, Irvine, CA, USA). For hepatic maturation, the Step-1 medium was replaced with Step-2 medium consisting of IMDM supplemented with 1 μM dexamethasone, 1% ITS, 20 ng/ml Oncostatin M (OSM, Peprotech), 20 ng/ml hHGF, and 10 uM Fasudil.
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Denuded blastocysts were placed onto mitomycin C (Sigma)-treated mouse embryonic fibroblast (MEF) feeder layers in mESC derivation medium: KODMEM (Gibco) containing 20% KOSR (Gibco), 1 mM L-glutamine (Gibco), 100 units/ml penicillin (Hyclone), 100 μg/ml streptomycin (Hyclone), 100 μM β-mercaptoethanol (Sigma), 100 μM nonessential amino acid, 10 μM fasudil (Adooq), 0.5 μM PD0325901 (PeproTech), 3 μM CHIR99021 (PeproTech), and 1000 units/ml LIF (Stemgent) under 5% CO2 at 37 °C in a humidified incubator9 (link),31 . The cell outgrowth was dissociated using trypsin-EDTA and seeded on the new MEF feeder layer. Established mESCs were passaged every 3–4 days using trypsin-EDTA for further experiments.
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For NCC differentiation, iPSCs were plated on a VTN-coated surface. After iPSCs reached approximately 30–40% confluence, the medium was switched to a neural crest induction E6 medium (Life Technologies) supplemented with 500 nM LDN19318 (Selleckchem, Houston, TX, USA), 100 nM BGJ398 (Selleckchem), and 10 μM fasudil (Adooq, Irvine, CA, USA). One day after differentiation, the medium was switched to E6 medium, supplemented with 1400 nM CHIR99021 (Peprotech), 100 ng/mL FGF2 (Peprotech), 250 nM SAG (Selleckchem), and 10 μM fasudil. The following day (day two), the medium was switched again to E6 medium supplemented with 100 nM BGJ398, 20 ng/mL BMP4 (Peprotech), and 10 μM fasudil. On day three, the medium was switched to E6 medium, supplemented with 10 ng/mL FGF2 and 10 μM fasudil. This medium was replaced each day for four days.
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EB formation was performed as described previously, with minor modifications (22 (link)). Dissociated iPSCs were seeded onto a StemFIT 3D culture dish (MicroFIT) and cultured in Essential 6 (Gibco) with 100 U/ml penicillin, 100 mg/ml streptomycin (GE Life Science), and 10 μM fasudil (Adooq) for two days. Aggregated cells were transferred to Petri dishes and cultured for six days using the same medium.
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Isolation of mouse embryonic fibroblasts (MEFs) was performed as previously described (Kang et al., 2014) .
MEFs were cultured with DMEM/F12 (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM glutamine (Life Technologies), 0.1 mM nonessential amino acids (Life Technologies), 0.1 mM 2-mercaptoethanol (2-ME, Sigma-Aldrich), 100 U/mL penicillin, 100 mg/mL streptomycin (GE Life Science) and 10 μM fasudil (Adooq) on gelatin-coated culture plates. The medium was changed once every three days. mESCs were cultured using mES medium; KODMEM supplemented with 20% Knockout Serum Replacement (KSR; Life Technologies), 1000 U/mL leukemia inhibitory factor (LIF; Millipore), 3 μM CHIR99021 (Peprotech), 0.5 μM PD0325901 (Peprotech), 2 mM glutamine, 0.1 mM nonessential amino acids, 0.1 mM 2-ME, 100 U/mL penicillin, and 100 mg/mL streptomycin on MEF feeder cells coated plates.
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All animals were cared for humanely in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (8th edition; Washington DC, National Academic Press, 2011). The protocol was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.
Male Sprague-Dawley rats (body weight, 310-340 g) were purchased from Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Animals were maintained on laboratory chow and housed in a specific pathogen-free room at a constant temperature (20-22°C) with 12 equal hours of light and dark exposure.
Fasudil was purchased from AdooQ Bioscience (Irvine, Calif) and was dissolved in 0.9% saline as a stock solution. Pentobarbital sodium was purchased from Sigma Aldrich (St. Louis, Mo).
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