Amicqui-1 were cultured on iMEF for at least 10 passages under two naïve conditions reported by Duggal et al., 2015 (link) and Zimmerlin et al., 2016 (link). Duggal medium is composed of hESC medium (DMEM/F-12, 20% KOSR, non-essential amino acids and mercaptoethanol) supplemented with 10 ng/mL FGF2, 1000 U LIF (Peprotech, 300–05), 1 µM PD0325901 (Tocris, 4192), 3 µM CHIR99021 (Tocris, 4423), 10 µM forskolin (Stemgent, 04–0025), and 50 ng/mL acid ascorbic (Sigma-Aldrich, A4544). Zimmerlin medium is constituted by hESC medium supplemented with 1000 U LIF, 1 µM PD0325901, 3 µM CHIR99021, and 4 µM XAV939 (Tocris, 3748). The passages were carried out every 3–5 days using Accutase. Cells in passage 7 were used for detection of naïve pluripotency markers by qPCR.
Pd0325901
Manufactured by Bio-Techne
Sourced in United Kingdom
PD0325901 is a small molecule inhibitor of the MEK1/2 enzymes. It functions by blocking the activation of the ERK1/2 signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Bio-Techne (17 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Glutamax, by Thermo Fisher Scientific (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (3 mentions)
Tryple, by Thermo Fisher Scientific (3 mentions)
B27 supplement, by Thermo Fisher Scientific (3 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
L alanyl l glutamine, by Thermo Fisher Scientific (2 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (2 mentions)
Rock inhibitor, by Bio-Techne (2 mentions)
Glial derived neurotrophic factor gdnf, by Thermo Fisher Scientific (2 mentions)
Smoothened agonist sag, by Merck Group (2 mentions)
Iwp 2, by Merck Group (2 mentions)
Iwp 2, by Thermo Fisher Scientific (2 mentions)
42 protocols using pd0325901
1
Naïve Pluripotency Marker Detection
2
Feeder-free mouse embryonic stem cell culture
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Pluripotent mouse embryonic stem cell lines R1 (Nagy et al., 1993 (link)) and E14tg2a (Kuehn et al., 1987 (link), Doetschman et al., 1987 (link)) were obtained from Neil Smyth, Southampton University, Southampton, UK. Cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM; life technologies, Paisley, UK, #31053-028) with 1% Penicillin/Streptomycin (PAA, Yeovil, UK, #P11-10) that was further supplemented with 15% KnockOut serum replacement, 1x MEM non-essential amino acids, 1x GlutaMax (all from life technologies, Paisley, UK, #10828-010, #11140-050 and #35050-038), 50 μM 2-mercaptoethanol (Sigma Aldrich, Gillingham, UK, #M6250). Leukaemia inhibitory factor (LIF), produced in house, was added at a saturating dilution of 1:1000. Cells were seeded on 0.1% gelatine (Sigma-Aldrich, Gillingham, UK, Cat. No. G1890) coated tissue culture plates pre-seeded with γ-irradiated MEF for routine culture. Throughout four subsequent passages prior to the start of the experiment, cells were cultivated in 0.1% gelatine coated tissue culture plates without additional MEF, and medium was additionally supplemented with a combination of 1 μM PD0325901 (Tocris bioscience, #4192) and 10 μM CHIR99021 (Reagents Direct, #27-H76). Cells were maintained at 37°C and 5% CO2 and routinely passaged every other day using Trypsin/EDTA (PAA, Yeovil, UK, #L11-003). Medium was replaced on a daily basis.
3
Maintenance of Murine and Rat ESCs
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R1 mESCs were cultured on 0.1% gelatin-coated dishes in Dulbecco’s modified Eagle’s medium (Gibco, catalog no. 11995065) containing 15% FBS (Hyclone, SH30396.03), leukemia inhibitory factor (LIF) (1000 IU/ml; Merck Millipore, ESG1107), penicillin (100 IU/ml) and streptomycin (100 μg/ml) (Merck Millipore, TMS-AB2-C), 2 mM GlutaMAX (Gibco, 35050061), 1% EmbryoMax nucleosides (Merck Millipore, ES-008-D), 0.1 mM nonessential amino acids (Gibco, 25-025-CIR), and 0.1 mM 2-mercaptoethanol (Gibco, 21985023). Rat ESCs were cultured on 0.1% gelatin-coated dishes in 2i/LIF medium [N2B27 medium supplemented with 1 μm of PD0325901 (Tocris, 41921), 3 μm of CHIR99021 (Tocris, 4423), and LIF (1000 IU/ml)].
4
Maintenance of Mouse Naive ES Cells
5
Cell Line Targeted Inhibition Assay
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NCI-60 cell lines were obtained from the National Cancer Institute (MTA No. 2702-09). Additional breast cancer cell lines were purchased from either the Korean Cell Line Bank (Seoul, Korea) for MDA-MB-453, HCC-1954, HCC-1937, BT-20, JIMT-1, HCC-1419, and HCC-38, or from the American Tissue Culture Collection (Manassas, VA, USA) for SKBR3, HCC-1569, and Au565.
The following targeted drugs were employed as targeted inhibitors: FAK inhibitor 14 (F14) for FAK, GW583340 for ERBB2, PHA665752 for MET, and PD0325901 for MAP2K2 from Tocris Bioscience (Bristol, UK); PD173074 for FGFR1 from Abcam (Cambridge, UK); c-MYC inhibitor II, c-MYC inhibitor for MYC, and Gefitinib for EGFR, and Podophyllotoxin for IGF1R from Sigma-Aldrich (St. Louis, MO, USA).
The following targeted drugs were employed as targeted inhibitors: FAK inhibitor 14 (F14) for FAK, GW583340 for ERBB2, PHA665752 for MET, and PD0325901 for MAP2K2 from Tocris Bioscience (Bristol, UK); PD173074 for FGFR1 from Abcam (Cambridge, UK); c-MYC inhibitor II, c-MYC inhibitor for MYC, and Gefitinib for EGFR, and Podophyllotoxin for IGF1R from Sigma-Aldrich (St. Louis, MO, USA).
6
Differentiation of iPSCs to Cortical Neurons
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Induced pluripotent stem cells were differentiated to neurons of cortical layer V and VI according to published protocols with minor modifications (Shi et al., 2012 (link); Rehbach et al., 2019 ). Briefly, iPSCs were seeded at a density of 3 × 105 cells per cm2 in E8 medium supplemented with 10 μM Y-27632 (Selleckchem). Neural induction was obtained by supplementation of SMAD inhibitors [10 μM SB431542 (Sigma-Aldrich) and 500 nM LDN-193189 (Sigma-Aldrich)] to 3N medium for 10 days. On day 10, cells were split in a 1:3 ratio and further expanded in 3N medium including 20 ng/ml FGF-2 for 2 days. Until day 27, cells were cultivated in 3N medium with media change every other day. On day 27, cells were dissociated using Accutase (Sigma-Aldrich) and replated at a density of ∼8 × 105 cells per cm2. The following day, 10 μM PD0325901 (Tocris) and 10 μM DAPT (Sigma Aldrich) was added to 3N medium with an additional media change at day 30. From day 32 onward 3N medium was changed every other day and RNA of cortical neurons was isolated at day 37 of differentiation.
7
Directed Differentiation of hPSCs to Cardiomyocytes
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hPSCs maintained on a Matrigel-coated surface in mTeSR1 were dissociated into single cells with Accutase (Life Technologies) at 37°C for 5 min and then seeded onto a Matrigel-coated cell culture dish at 50,000 cell/cm2 in mTeSR1 supplemented with 5 μM ROCK inhibitor Y-27632 (Selleckchem) (day −3) for 24 hr. Cells were then cultured in mTeSR1, changed daily. At day 0, cells were treated with 6–10 μM CHIR99021 (Selleckchem) for 2 days in LaSR basal medium, which consists of Advanced DMEM/F12, 2.5 mM GlutaMAX, and 60 μg/ml ascorbic acid (Sigma, A8960). After 2 days, CHIR99021-containing medium was aspirated and cells were maintained in LaSR basal medium without CHIR99021 for 3–4 additional days. PD0325901 was purchased from Tocris Bioscience.
8
Characterization of Genetically Modified ES Cells
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The W4 ES cell line was provided by the Rockefeller University Core Facility and W4 p53 KO ES cell line was previously generated by CRISPR/Cas9 technology in our lab [38 (link)]. Cells were routinely cultured in 2i/LIF ES medium consisting of DMEM, Glutamax (2 mM), MEM NEAA (100 mM), 2-mercaptoethanol (0.1 mM), penicillin (100 U/mL), streptomycin (100 mg/mL) and FBS (15%, Gibco, Paisley, UK), LIF, PD0325901 (1 μM, Tocris, Bristol, UK) and CHIR 99,021 (3 μM, Tocris). Cells were plated on bovine gelatin (0.1%, Sigma, St. Louis, MO, USA) coated dishes at 37 °C in a 5% CO2 (v/v) incubator and passaged every three days. To induce HO-1 expression, cells were pre-treated or treated with hemin (20 μM, Sigma) during 20 h. Cell viability and proliferation were evaluated using MTT (Sigma) and Crystal Violet. To inhibit protein synthesis cells were treated with cycloheximide (10 μM, Sigma) during 2 or 7 h. To induce differentiation cells were cultured in the absence of LIF and 2i for 48 h. To evaluate the effect of H2O2 on HO-1 induction cells were exposed to H2O2 (100 μM) during 4 h.
9
Directed Differentiation of hPSCs to AME-E and AME-L
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For differentiation to AME-E-like cells, partially primed hPSC were dissociated to single cells using TrypLE Express and counted. The cells were plated to Geltrex-coated tissue culture plates at a seeding density of 1x105/cm2 in differentiation medium with 10μM ROCK inhibitor, and further cultured for 5 days. Differentiation medium was prepared as following: N2B27 basal medium, 1μM PD0325901 and 1μM A8301 (Cat. 2939, Tocris Bio-Techne). 100nM LDN193189 (alternative name DM3189, Cat. 1509, Axon Medchem) or 20ng/ml BMP4 (Miltenyi Biotec) were optionally added to the medium. The medium was changed daily. When the spheres appeared, the medium was refreshed by careful exchange of half volume of the medium, and the volume of the medium per well was increased.
Differentiation to TE-like cells was performed according to Guo et al. (2021) (link) and Io et al. (2021) (link). For differentiation to AME-L-like cells, primed hPSC were dissociated to single cells using TrypLE Express and counted. The cells were plated to Geltrex-coated tissue culture plates at a seeding density of 1x105/cm2 in differentiation medium with 10μM ROCK inhibitor, and further cultured for 5 days. Differentiation medium was prepared as following: N2B27 basal medium, 1μM PD0325901, 1μM A8301 (Cat. 2939, Tocris Bio-Techne) and 20ng/ml BMP4 (Miltenyi Biotec). The medium was changed daily.
Differentiation to TE-like cells was performed according to Guo et al. (2021) (link) and Io et al. (2021) (link). For differentiation to AME-L-like cells, primed hPSC were dissociated to single cells using TrypLE Express and counted. The cells were plated to Geltrex-coated tissue culture plates at a seeding density of 1x105/cm2 in differentiation medium with 10μM ROCK inhibitor, and further cultured for 5 days. Differentiation medium was prepared as following: N2B27 basal medium, 1μM PD0325901, 1μM A8301 (Cat. 2939, Tocris Bio-Techne) and 20ng/ml BMP4 (Miltenyi Biotec). The medium was changed daily.
10
Culturing Naïve Pluripotent Mouse ES Cells
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Mouse ES cells were cultured in a medium composed of DMEM (Gibco), 2 mM Glutamax (Gibco), 100 mM MEM nonessential amino acids (Gibco), 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco), supplemented with 15% FBS (Gibco), LIF and 2i (1 μM PD0325901 and 3 μM CHIR99021, Tocris). The use of these inhibitors allows culturing ES cells preserving naïve pluripotency [114 (link)].
Cells were maintained on 0.1% gelatin coated dishes at 37 °C in a 5 % CO2 (v/v) incubator and passaged every 3 days using trypsin (Gibco) and routinely assessed for mycoplasma contamination by genomic DNA extraction and PCR analysis.
The experiments were performed using two cell lines: the mouse ES cell line W4 provided by the Rockefeller University Core Facility and the YPet-OCT4 ES cell line, previously generated in our laboratory from the same W4 cell line [61 (link)]. The YPet-OCT4 cell line expresses the TF OCT4 fused to the fluorescent protein YPet in a doxycycline-inducible manner. Cells were incubated with 5 μg/ml doxycycline for 48 h prior to imaging experiments.
Cells were maintained on 0.1% gelatin coated dishes at 37 °C in a 5 % CO2 (v/v) incubator and passaged every 3 days using trypsin (Gibco) and routinely assessed for mycoplasma contamination by genomic DNA extraction and PCR analysis.
The experiments were performed using two cell lines: the mouse ES cell line W4 provided by the Rockefeller University Core Facility and the YPet-OCT4 ES cell line, previously generated in our laboratory from the same W4 cell line [61 (link)]. The YPet-OCT4 cell line expresses the TF OCT4 fused to the fluorescent protein YPet in a doxycycline-inducible manner. Cells were incubated with 5 μg/ml doxycycline for 48 h prior to imaging experiments.
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