iPSCs were manually dissected from MEF, then grown in suspension culture for 4 days in EB medium supplemented with 2 μM dorsomorphin (R&D Systems, 3096; Minneapolis, MN, USA) and 5 μM SB431542 (R&D Systems, 1614). Medium was then changed to neural induction medium consisting of DMEM/F12, 1 × N2 supplement (Invitrogen, 17502-048), 1 × NEAA, 2 mM glutamax, 0.1 mM β-mercaptoethanol, 2 μM dorsomorphin, 5 μM SB431542 and 20 ng ml−1 FGF2 for an additional 3 days in suspension. Neurospheres were then planted on laminin (Sigma-Aldrich, L2020)-coated culture dishes for rosette formation. Rosettes were manually picked 2–3 times, dissociated in Accutase then re-plated on poly-D-ornithine-laminin-coated substrates for neuronal differentiation in medium composed of neural basal medium (Invitrogen, 21103–049), 1 × B27 supplement (Invitrogen, 17504–044), 1 mM glutamine, 1% NEAA and 1 × Penicillin/Streptomycin for an additional 4, 8 or 12 weeks. Medium was changed every other day. To determine whether dorsal–ventral fate could be respecified, cells were grown with the Smoothened agonist (Hedgehog pathway activator) purmorphamine (1 μm; Cayman Chemical, Ann Arbor, MI, USA, 10009634), or with the dorsalizing agent lithium chloride (LiCl; 1 mM ; Sigma-Aldrich). Controls were exposed to DMSO (carrier) alone.42 (link),43 (link)
Sb431542
Manufactured by Thermo Fisher Scientific
Sourced in United States
SB431542 is a cell culture reagent that functions as a selective inhibitor of the Transforming Growth Factor-Beta (TGF-β) signaling pathway. It acts by blocking the activation of the TGF-β type I receptor, ALK5. This product is intended for research use only and not for use in diagnostic or therapeutic procedures.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Thermo Fisher Scientific (3 mentions)
Sk br 3, by National Collection of Authenticated Cell Cultures (3 mentions)
Mda mb 231, by National Collection of Authenticated Cell Cultures (3 mentions)
Mcf 7, by National Collection of Authenticated Cell Cultures (3 mentions)
A3160801, by Thermo Fisher Scientific (3 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Purmorphamine, by Cayman Chemical (2 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Y27632, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Keygen Biotech (2 mentions)
Bay 11 7085, by Enzo Life Sciences (2 mentions)
Cc401, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Dorsomorphin, by R&D Systems (1 mentions)
Sb431542, by R&D Systems (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Cellstart, by Thermo Fisher Scientific (1 mentions)
24 protocols using sb431542
1
Neuronal Differentiation from iPSCs
2
Differentiation of iPSCs to RPE
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The iPSCs were differentiated to RPE according to the protocol established by Dr. Osakada (Osakada et al., 2009 (link)). The iPSCs clumps were first incubated in human ES cell culture medium supplemented with 10 μM Y-27632 (WAKO), 5 μM SB431542 (Sigma–Aldrich) and 3 μM CKI-7 (Sigma–Aldrich) for 1 day. The cells were incubated in a differentiation medium (Glasgow minimum essential medium [GMEM; Invitrogen], 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 0.1 mM 2-mercaptoethanol) containing 20% knockout serum replacement (KSR; Invitrogen) for 4 days, then in 15% KSR-containing differentiation medium for 6 days, and finally in 10% KSR-containing differentiation medium for 11–40 days. Y-27632 (10 μM), SB431542 (5 μM) and CKI-7 (3 μM) were added to the differentiation medium for the first 13 and 19 days, respectively. Partially differentiated cells were dissociated and incubated on non-adhesive dishes (Corning) in RPE maintenance medium (DMEM:F12 [7:3] supplemented with B-27 supplement [Invitrogen] and 2 mM l-glutamine [Invitrogen]) for 10 days. The resulting RPE cell aggregates were isolated and replated on CELLstart- (Invitrogen) coated dishes in RPE maintenance medium supplemented with 0.5 μM SB431542 and 10 ng/ml bFGF. The medium was changed every 2–3 days. Thereafter, RPE cells formed compact monolayers and re-pigment, typically 90–120 days.
3
Differentiation of hPSCs into Neural Progenitor-like Cells
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On day 0, hPSCs were differentiated in N2 medium [500 mL DMEM/F12 (1:1) (Gibco, Cat # 11320-033), 5 mL Glutamax (Gibco, Cat # 35050-061), 7.5 mL Sucrose (20%, SIGMA, Cat # S0389), 5 mL N2 supplement B (StemCell Technologies, Cat # 07156)] supplemented with SB431542 (10 μM, Tocris, Cat # 1614), XAV939 (2 μM, Stemgent, Cat # 04-00046) and LDN-193189 (100 nM, Stemgent, Cat # 04-0074) along with doxycycline hyclate (2 μg.mL-1, Sigma, Cat # D9891) with Y27632 (5 mM, Stemgent, Cat # 04-0012). Day 1 was a step-down of small molecules, where N2 medium was supplemented with SB431542 (5 μM), XAV939 (1 μM) and LDN-193189 (50 nM) with doxycycline hyclate (2 μg.mL-1) and Zeocin (1 μg.mL-1, Invitrogen, Cat # 46-059). On day 2, N2 medium was supplemented with doxycycline hyclate (2 μg.mL-1) and Zeocin (1 μg.mL-1). Starting on day 2 human induced neural progenitor-like cells were harvested with Accutase (Innovative Cell Technology, Inc., Cat # AT104-500) and re-plated at 15,000 cells.cm-2 in Astrocyte Medium (ScienCell, Cat # 1801) with Y27632 (5 mM) on geltrex coated plates. Cells were maintained for > 30 days in Astrocyte Medium (ScienCell, Cat # 1801).
4
Vascular Differentiation of Human iPSCs
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HiPSCs were differentiated using a modified STEMdiff APEL-Li Vascular differentiation method previously described in Park et al., 2020 (41 (link)). Briefly, on day 1, E8 medium was replaced with APEL-2Li medium (StemCell Technologies, cat # 5271) supplemented with Activin A (25 ng/mL, Fisher Scientific, cat # 50398465), VEGF (50 ng/mL, Fisher Scientific, cat #501628212), BMP4 (30 ng/mL, Fisher Scientific, cat #50398981), and CHIR99021 (1.5 μM, Fisher Scientific, cat #NC0984214) for the first 2 days. Subsequently, APEL-Li supplemented with VEGF (50 ng/mL) and SB431542 (10 μM, Fisher Scientific, cat # NC0548146) were used and replaced previous differentiation medium every 2 days until cell population reached maximum expression of CD31+ percentage as determined by Flow Cytometry analysis. CD31+ ECs were isolated using CD31+ Dynabeads (Life Technologies, cat # 11155D). ECs were expanded on fibronectin coated plates in Endothelial Growth Medium-2 (Lonza, cat # CC-3162) for at least 7 to 9 days. HiPSC-ECs at 60% confluency were treated with 30 µM of heme (Sigma-Aldrich, Inc., Saint Louis MO, cat # H9039) or volume-equivalent of vehicle (DMSO, ThermoFisher, cat # 85190) for 24 hours.
5
Heat Shock and Pharmacological Inhibitor Protocol
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For heat shock, fish were immersed in a water bath at 37°C for 1 hr before returning to system water at 28°C. For extended periods of heat shock, this was repeated every 6 hr. For inhibition of Tgfb signaling we used two different Tgfb receptor 1 (Alk 5) inhibitors, SB431542 and SB505124 (Fisher Scientific) and to inhibit Notch signaling we used RO4929097 (Cayman). PP2A was inhibited with okadaic acid (Cell Signaling Technology) and p38 MAPK was inhibited with PD169316 (Cayman Chemical). Pharmacological reagents were prepared in DMSO as a 10 mM stock and diluted 1/200 in fish water for immersion or PBS for intravitreal injections. Control fish were treated with vehicle.
6
Neural Differentiation Cocktail Protocol
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Cells were primed in the NM for 3 days before the treatment with chemical cocktails S/C/D or morphogens S/F8/F2. The dosage are as follows: 5 µM SB431542 (Peprotech, East Windsor, NJ, USA; 3014193), 1.5 µM CHIR99021 (Peprotech, East Windsor, NJ, USA; 2520691), 5 µM DAPT (Peprotech, East Windsor, NJ, USA; 2088055), 250 ng/mL SHH (Peprotech, East Windsor, NJ, USA; 100-45-25), 100 ng/mL FGF8 (Peprotech, East Windsor, NJ, USA; 100-25A-25), and 50 ng/mL FGF2 (Peprotech, East Windsor, NJ, USA; 100-18B-10). The NM was changed every 2–3 days, and the fresh chemicals S/C/D or morphogens S/F8/F2 were added to the NM, respectively (Figure 3 A and Figure 4 A).
7
Naïve Conditions for Deriving Trophoblast Stem Cells
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For short-term pre-exposure, hPSCs were exposed for 4 days to naïve conditions. Naïve cocktails used were: CDK8/19i cocktail [0.4 μM of CNIO-CDK8/19 inhibitor and 20 ng/mL hrLIF (Peprotech)], as reported [20 (link)], or the 2i-based cocktail PXGL [1 μM PD0325901 (Axon Medchem), 2 μM Gö6983 (Selleckchem), 2 μM XAV939 (Selleckchem) and 20 ng/mL hrLIF (Peprotech)] [8 (link)]. TSCs derivation was performed as previously reported by Kojima and colleagues [23 (link)]. Naïve and primed hPSCs were single-cell dissociated by TrypLE Express, and 0.5 × 106 cells were seeded in a 6-well plated pre-coated with 5 mg/mL Collagen IV (#354233, Corning) and cultured in TS medium [DMEM/F12 (D6421, Sigma), 0.1 mM 2-mercaptoethanol (Gibco), 0.2% FBS (Gibco), 0.3% BSA (Sigma), 1% ITS-X (Gibco), 1.5 μg/mL L-ascorbic acid (Sigma), 50 ng/mL EGF (PeproTech), 2 μM CHIR99021 (Axon Medchem), 0.5 μM A83-01 (Tocris, Bristol, UK), 1 μM SB431542 (PeproTech), 0.8 mM VPA (Sigma) and 5 μM Y-27632 (Selleckchem)] [24 (link)]. Cells were passaged every 5–7 days in single cells using TrypLE Express on pre-coated Collagen IV plates.
8
Investigating Breast Cancer Cell Lines
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SK-BR-3, MCF-7 and MDA-MB-231 cell lines used in this study were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MCF-10A (normal human mammary epithelial cell line) purchased from KeyGEN Biotech Company (Nanjing, China) was used as the control cell line. All cell lines were grown in Dulbecco’s Modified Eagle’s medium (DMEM, KeyGEN) supplemented with 10% fetal bovine serum (A3160801, Gibco) and 1% penicillin–streptomycin. Cell culture was accomplished in a humidified incubator containing 5% CO2 at 37 °C. Cell passage was performed when the confluence reached to 90%. Reagents used in this study, including TGF-β1, TGF-β type I Receptor inhibitor SB431542, Notch pathway inhibitor FLI-06, were separately purchased from PeproTech (#100–21, USA), MedChemExpress (HY-10431, USA) and MedChemExpress (HY-15860, USA).
9
Differentiation of hiPSCs into Dopaminergic Neurons
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Neuronal differentiation of human iPSCs was induced as previously described (Arioka et al., 2018 (link)). Briefly, iPSCs were pretreated with 3 µM SB431542 (Cayman, Ann Arbor, MI, United States), 3 µM CHIR99021 (Cayman), and 3 µM dorsomorphin (Sigma, Burlington, MA, United States) for 1 week (day 0–7) and dissociated into single cells by incubation with TripLE Select (Gibco) for 5 min. The cells were cultured in neurosphere medium containing MHM [DMEM/F12, supplemented with 1× N2 (Gibco), 0.6% glucose (Sigma), 5 mM HEPES (Sigma), 100 units/mL penicillin and 100 μg/mL streptomycin, 1× B27 (Gibco), 20 ng/mL bFGF, 10 ng/mL hLIF (Nacalai, Kyoto, Japan), 10 µM Y-27632 (Cayman), 3 µM CHIR99021, 2 μM SB431542, 100 ng/mL FGF8b (Peprotech, Cranbury, NJ, United States), and 1 µM purmorphamine (Cayman) for 1 week (day 7–14)]. On day 14, neurospheres were passaged by dissociation into single cells in the same manner as primary neurosphere formation. For terminal differentiation into dopaminergic neurons, secondary neurospheres were dissociated 1 week after passage (day 21) and plated onto poly-L-ornithine/laminin/fibronectin-coated dishes at the density of 3 × 104 cells/cm2 in dopaminergic neuron medium [MHM supplemented with B27, 10 µM DAPT (Cayman), 20 ng/mL BDNF (Peprotech), 20 ng/mL GDNF (R&D), 0.2 mM ascorbic acid (Sigma), 1 ng/mL TGF-β3 (R&D), and 0.5 mM dbcAMP (Sigma)].
10
Differentiated Colonic Organoid Monolayers
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Human colonoids were prepared from biopsy samples of adult healthy individuals at the Digestive Diseases Research Center of the Washington University, School of Medicine and were grown as previously described (49 (link), 50 (link)). Briefly, isolated colonoids were thawed and plated (in 24-well plates) in Matrigel (BD Biosciences) droplets (15 μl) followed by incubation at 37 °C with conditioned media (a 1:1 mixture of the L-WRN cell line and primary culture media [Advanced DMEM/F12; Invitrogen] supplemented with fetal bovine serum [20%], l -glutamine [2 mM], penicillin [100 U/ml], streptomycin [100 μg/ml], Stemolecule Y27632 [10 μM; Reprocell], and SB 431542 [10 μM; Peprotech]). To obtain polarized differentiated colonoid monolayers, cells were plated at density of 5 × 104 cells/well onto 24-well cell culture inserts (Corning) coated with type IV human collagen (Sigma) and then grown for 4 days prior to induction of differentiation by addition of differentiation media (5% conditioned media added with only Y-27632 inhibitor). Differentiated colonoid monolayers were then exposed as indicated to normoxia and hypoxia conditions as mentioned previously.
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