Fibroblast medium: DMEM (ThermoFisher), 10% fetal bovine serum (FBS, Hyclone), 1% non-essential amino acids (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 55 μM β-mercaptoethanol (ThermoFisher) and 1 mM sodium pyruvate (ThermoFisher). Naive medium (t2iLGoY)19 (link): 50:50 mixture of DMEM/F12 (ThermoFisher) and neurobasal medium (ThermoFisher), supplemented with 2 mM l -glutamine (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 10 ng ml−1 human leukaemia inhibitory factor (made in-house), 250 μM l -ascorbic acid (Sigma), 10 μg ml−1 recombinant human insulin (Sigma), 1 μM PD0325901 (Miltenyi Biotec), 1 μM CHIR99021 (Miltenyi Biotec), 2.5 μM Gö6983 (Tocris), 10 μM Y-27632 (Abcam). Primed hiPS cell medium (KSR/FGF2): DMEM/F12 (ThermoFisher), 20% knockout serum replacement (KSR) (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 50 ng ml−1 recombinant human FGF2 (Miltenyi Biotec), 1% penicillin-streptomycin (ThermoFisher). Primed hiPS cell medium (Essential 8 (E8)): 10 ml of E8 supplement (Gibco) to 500 ml medium basal (Gibco), supplemented with 1% penicillin-streptomycin (Gibco).
G 6983
Manufactured by Bio-Techne
Sourced in United States, United Kingdom
Gö6983 is a potent and selective inhibitor of protein kinase C (PKC) isoforms. It effectively blocks the catalytic activity of PKC and is commonly used as a research tool to study PKC-dependent signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (4 mentions)
G 6976, by Bio-Techne (4 mentions)
B27 supplement, by Thermo Fisher Scientific (3 mentions)
Phorbol myristate acetate (pma), by Bio-Techne (3 mentions)
E8 supplement, by Thermo Fisher Scientific (2 mentions)
N2 supplement, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Y 27632, by Abcam (2 mentions)
Recombinant human insulin, by Merck Group (2 mentions)
L ascorbic acid, by Merck Group (2 mentions)
Medium basal, by Thermo Fisher Scientific (2 mentions)
Recombinant human fgf2, by Miltenyi Biotec (2 mentions)
22 protocols using g 6983
1
Fibroblast and Induced Pluripotent Stem Cell Media Formulations
2
Maintenance of Naive Human Pluripotent Stem Cells
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Naïve hPSC were maintained on irradiated mouse embryonic fibroblasts (MEF) in PDLGX medium prepared as following: N2B27 supplemented with 1μM PD032590, 10ng/ml human LIF (both from Cambridge Stem Cell Institute facility), 2μM Gö6983 (Tocris Bio-Techne, Cat. 2285), and 2μM XAV939 (Tocris Bio-Techne, Cat. 3748), as described previously (Rostovskaya et al., 2019 ). N2B27 basal medium was prepared as following: Neurobasal (Cat. 21103049, ThermoFisher Scientific) and DMEM/F12 (Cat. 31331093, ThermoFisher Scientific) in the ratio 1:1; 0.5% N2 (Cat. 17202048, ThermoFisher Scientific), 1% B27 (Cat. 17504044, ThermoFisher Scientific), 2mM L-glutamine (Cat. 25030024, ThermoFisher Scientific), 100μM 2-mercaptoethanol (Cat. M7522, Sigma-Aldrich). Geltrex (A1413302, ThermoFisher Scientific) was added at a concentration 0.5μl/ml to the culture medium during re-plating. Naïve hPSC were passaged using TrypLE Express (Cat. 12604021, ThermoFisher Scientific) as single cells. 10μM ROCK inhibitor (Y-27632, Cat. 688000, Millipore) was added for 24 hours after passaging.
Conventional primed H9 hPSC (WA09, WiCell) were cultured in mTeSR-E8 media (Cat. 05990, STEMCELL Technologies) (Chen et al., 2011 (link)) on Geltrex pre-coated plates and passaged using 0.5mM EDTA in PBS. All cells were cultured in a humidified incubator with 5% O2 and 5% CO2 at 37°C.
Conventional primed H9 hPSC (WA09, WiCell) were cultured in mTeSR-E8 media (Cat. 05990, STEMCELL Technologies) (Chen et al., 2011 (link)) on Geltrex pre-coated plates and passaged using 0.5mM EDTA in PBS. All cells were cultured in a humidified incubator with 5% O2 and 5% CO2 at 37°C.
3
Xeno-free Culture of Human iPSCs
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The study was approved by the Ethical Committee of the CHA University Bundang CHA Hospital, Republic of Korea (application number: KNC12005). Human adult dermal fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in DMEM (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Invitrogen) and 1x penicillin/streptomycin (P/S) (all from Invitrogen, Carlsbad, CA, USA).
Human iPS cells were cultured on vitronectin XF (Primorigen Biosciences, Madison, USA) coated culture dishes using our recently established xeno-free/feeder-free hPSC culture medium with minor modifications [17 (link)]. Briefly, the medium consisted of DMEM/F12, 15% KnockOut SR XenoFree CTS, 1x nonessential amino acids (NEAA), 1x GlutaMAX, 0.1 mM β-mercaptoethanol, 1x P/S (all from Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF) (CHA Biotech Co., Daejeon, Korea), 10 nM trichostatin A (TSA) (Sigma-Aldrich, St. Louis, MO, USA), 5 uM Gö6983 (Tocris, Ellisville, MO, USA), and 1 mM dorsomorphin dihydrochloride (Tocris).
Human iPS cells were cultured on vitronectin XF (Primorigen Biosciences, Madison, USA) coated culture dishes using our recently established xeno-free/feeder-free hPSC culture medium with minor modifications [17 (link)]. Briefly, the medium consisted of DMEM/F12, 15% KnockOut SR XenoFree CTS, 1x nonessential amino acids (NEAA), 1x GlutaMAX, 0.1 mM β-mercaptoethanol, 1x P/S (all from Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF) (CHA Biotech Co., Daejeon, Korea), 10 nM trichostatin A (TSA) (Sigma-Aldrich, St. Louis, MO, USA), 5 uM Gö6983 (Tocris, Ellisville, MO, USA), and 1 mM dorsomorphin dihydrochloride (Tocris).
4
Aflatoxin Detection Assay Protocol
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Unless indicated, all reagents and solutions were as previously described40 (link)43 (link)77 (link)78 (link). Fluo-4 and DAF-FM were from Invitrogen (Grand Island, NY). Aflatoxins B1 (AFB1) and B2 (AFB2) were from Cayman (Ann Arbor, MI). Gö6983, BIIE-0246, [Leu31,Pro34]-NPY, NPY-(16–36), antagonist G, and calphostin C were from Tocris (Minneapolis, MN USA). All other reagents were from Sigma-Aldrich (St. Louis, MO USA). Stock solutions of aflatoxin were 10 mM in DMSO. Working solutions (10, 1, and 0.1 μM) contained 0.1%, 0.01%, and 0.001% DMSO, respectively, and were made up immediately before use; activities of aqueous solutions were observed to be markedly reduced after ~1–2 hr at room temp. Anti-aflatoxin antibodies (recognizing AFB1, AFB2, and AFG) were from Thermo Scientific (MA-7386) and Sigma Aldrich (A9555).
5
Culturing Naive Human Pluripotent Stem Cells
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Naive hPSCs (H9 hESCs, WIBR2-MGT (converted from WIBR2 29M-GP26-TN9 hESCs) and Sigma hiPSCs) were cultured on MMC-MEF feeders in 5% O2 and 5% CO2 incubator under humidified conditions at 37°C. All naive hPSCs were cultured in PXGL medium (Bredenkamp et al., 2019b (link)) which consists of 1:1 DMEM/F12 and Neurobasal, 0.5% N2-supplement, 1% B27-Supplement, 2 mM L-Glutamine, 0.1 mM β-mercaptoethanol, 1x penicillin-streptomycin, 1 μM PD0325901 (Axon Medchem), 2 μM XAV939 (Sigma-Aldrich, X3004), 2 μM Gö6983 (Tocris, 2285), 20 pg/mL human LIF (PeproTech, 300-05) and 10 μM Y-27632 (Tocris, 1254). Naive hPSCs were passaged every 4–5 days in a ratio 1:2 or 1:3 by single-cell dissociation with Accutase (Sigma-Aldrich, A6964-100 ML) followed by filtering through a 40 μm cell strainer (Corning, 352340).
6
Evaluation of Novel Signaling Modulators
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Tamoxifen citrate, ceramide, MPP dihydrochloride, G-1, Gö 6983, Gö 6976, PKCζ-PS and DL-PDMP were all obtained from Tocris Bioscience (Bristol, UK). Myriocin was obtained from Cayman Chemical (Ann Arbor, MI). Unless otherwise noted, all other chemicals were obtained from Sigma Aldrich.
7
Maintenance and Passaging of Naïve and Primed Human Pluripotent Stem Cells
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All hPSC lines were maintained at 5% O2 and 5% CO2 at 37°C in a humidified incubator. Naïve hPSCs were cultured in N2B27 media composed of 1:1 DMEM/F12 and Neurobasal medium supplemented with 0.5× B-27, 0.5× N-2, 2 mM L-glutamine, 50 U/ml and 50 µg/ml penicillin-streptomycin and 0.1 mM β-mercaptoethanol (all ThermoFisher Scientific) and with 2 μM Gö6983 (Tocris), 1 μM PD0325901, 1 μM CHIR99021 and 20 ng/ml human LIF (all Wellcome-MRC Cambridge Stem Cell Institute) for t2iLGö medium (Takashima et al., 2014 (link)), or with 1 μM PD0325901, 2 μM Gö6983, 20 ng/ml human LIF and 2 µM XAV939 (Cell Guidance Systems) for PXGL medium (Bredenkamp et al., 2019b (link); Rostovskaya, 2022 (link); Rostovskaya et al., 2019 (link)). Naïve hPSCs were grown either on irradiated MF1 mouse embryonic fibroblasts (MEFs) (Wellcome-MRC Cambridge Stem Cell Institute) on plates pre-coated with 0.1% gelatin (Sigma-Aldrich) or in feeder-free conditions using Geltrex Matrix (ThermoFisher Scientific) added to medium at a 1:300 dilution. Naïve hPSCs were passaged by 5 min incubation at 37°C with Accutase (BioLegend). Primed hPSCs were cultured on plates pre-treated with 5 µg/ml Vitronectin (ThermoFisher Scientific) in mTeSR Plus medium (STEMCELL Technologies) and passaged by 5 min incubation at room temperature with 0.5 mM EDTA in PBS.
8
Cerebellar Slice Culture Preparation
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Slice cultures were prepared as described previously (Gugger et al., 2012 (link)). Briefly, mice were decapitated at postnatal day 8 (P8), their brains were aseptically removed, and the cerebellum was dissected in ice-cold preparation medium: MEM, 1% Glutamax (Invitrogen), pH 7.3. Sagittal sections (350 µm thickness) were cut on a McIllwain tissue chopper under aseptic conditions. Slices were separated, transferred onto permeable membranes (Millicell-CM, Millipore), and incubated on a layer of neurobasal medium: Neurobasal A medium (Invitrogen) supplemented with B27 supplement (Invitrogen) and Glutamax (Invitrogen), pH 7.3, in a humidified atmosphere with 5% CO2 at 37°C. The medium was changed every 2-3 d; 300 nm PMA (Tocris Bioscience) was added for PKC activation 24 h before slices were fixed; 10 μm Gö6983 (Tocris Bioscience) was added to the medium at each medium change for PKC inhibition, starting at 3 DIV (DIV3). Slices were kept in culture for a total of 7 d and analyzed with Western blot and immunohistochemical staining. To monitor protein degradation, slices were treated with 50 µg/ml cycloheximide (Sigma Millipore) or 10 μm MG132 (Sigma Millipore) or both at DIV7, and protein was extracted at DIV8.
9
Fibroblast and Induced Pluripotent Stem Cell Media Formulations
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Fibroblast medium: DMEM (ThermoFisher), 10% fetal bovine serum (FBS, Hyclone), 1% non-essential amino acids (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 55 μM β-mercaptoethanol (ThermoFisher) and 1 mM sodium pyruvate (ThermoFisher). Naive medium (t2iLGoY)19 (link): 50:50 mixture of DMEM/F12 (ThermoFisher) and neurobasal medium (ThermoFisher), supplemented with 2 mM l -glutamine (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 0.5% N2 supplement (ThermoFisher), 1% B27 supplement (ThermoFisher), 1% penicillin-streptomycin (ThermoFisher), 10 ng ml−1 human leukaemia inhibitory factor (made in-house), 250 μM l -ascorbic acid (Sigma), 10 μg ml−1 recombinant human insulin (Sigma), 1 μM PD0325901 (Miltenyi Biotec), 1 μM CHIR99021 (Miltenyi Biotec), 2.5 μM Gö6983 (Tocris), 10 μM Y-27632 (Abcam). Primed hiPS cell medium (KSR/FGF2): DMEM/F12 (ThermoFisher), 20% knockout serum replacement (KSR) (ThermoFisher), 1 mM GlutaMAX (ThermoFisher), 0.1 mM β-mercaptoethanol (ThermoFisher), 1% non-essential amino acids (ThermoFisher), 50 ng ml−1 recombinant human FGF2 (Miltenyi Biotec), 1% penicillin-streptomycin (ThermoFisher). Primed hiPS cell medium (Essential 8 (E8)): 10 ml of E8 supplement (Gibco) to 500 ml medium basal (Gibco), supplemented with 1% penicillin-streptomycin (Gibco).
10
Structural Plasticity in Neurons
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All pharmacological experiments were performed in a paired manner for each neuron. Structural plasticity was induced in spines before and after drug application in the same neuron. 1NMPP1 (Santa Cruz, WD 1 μM) was prepared as a 10 mM stock in DMSO and then diluted to 500x in a solubilisation buffer (0.9% NaCl and 2.5% Tween-20). U73122 and U73343 (Tocris, μM) WD 10 (Gö6983 (Tocris, 1 μM WD), Phorbol 12,13-dibutyrate (PdBu, Tocris, WD 1 μM), Edelfosine (Tocris, WD 1 μM), NMDA (Tocris, WD 20 μM), MNI Caged Glu (Tocris, 4 mM), APV (Sigma, WD 50 μM), S-MCPG (Tocris, WD 250 μM), NPS 2390 (Tocris, WD 20 μM) were stored as recommended by supplier and diluted into aCSF to the working dilutions (WD) listed.
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