H9 hESCs were transferred to vitronectin-coated 4-well plates in N2B27 medium (1:1 mix of neurobasal medium and DMEM/F12 medium, 1% insulin/transferrin/selenium, 1 × N2 supplement, 1 × retinol-free B27 supplement, 0.3% glucose, and 1% p/s (Thermo Fisher Scientific)) for 11 days of differentiation. As previously described [35 (link)], for rostral neural progenitor cell (NPC) differentiation, SB431542 (SB, 10 µM; Tocris, Bristol, UK) and LDN193189 (LDN, 0.1 µM; STEMCELL Technologies) were added to the media for 4 days. The cells were further cultured with SB, LDN, and FGF2 (20 ng/ml; STEMCELL Technologies) until day seven. Between day 7 and 11, the cell media was only supplemented with FGF2 (20 ng/ml). For caudal NPC differentiation, SB431542 (10 µM) and CHIR99021 (CHIR; 3 µM; STEMCELL Technologies) were added to the media from days zero to four. The cells were further cultured from days 4 to 11 in the absence of SB and CHIR, but with FGF2 (20 ng/ml). RNA was harvested at days 0 (before differentiation), 4, and 11.
Chir99021 chir
Manufactured by STEMCELL
Sourced in Canada
CHIR99021 (CHIR) is a small molecule inhibitor that selectively blocks glycogen synthase kinase 3 (GSK3) activity. It acts as an important regulator in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (2 mentions)
Sb431542, by STEMCELL (1 mentions)
Sb431542, by Bio-Techne (1 mentions)
Fibroblast growth factor 2 (fgf2), by STEMCELL (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Activin a, by Bio-Techne (1 mentions)
Rpmi b27 medium, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Embryonic stem cell qualified fbs, by Thermo Fisher Scientific (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Iwp 2, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium f12, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
5 protocols using chir99021 chir
1
Rostral and Caudal NPC Differentiation from hESCs
2
Directed Differentiation of hESCs to Endoderm
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We used a modified protocol based on D’Amour et al. and Sui et al. adapted to LN521 coating32 (link),34 (link). Briefly, hESCs were seeded at a density of 4 × 104 cells per cm2 and the differentiation was started 1–2 days later once the cells were 50–60% confluent. Cells were differentiated for 24 h in RPMI 1640 Medium with GlutaMAX and supplemented with 2% B27 supplement (both from Thermo Fisher Scientific), 3 µM CHIR99021 (CHIR, STEMCELL Technologies) and 100 ng/mL Activin A (Biotechne). After the 24-h mesendoderm specification, the cells were incubated for an additional 48 h in the same differentiation medium but without CHIR. When indicated, the DE differentiation conditions were modified: either 1.5, 3, 4.5 or 9 µM CHIR was used during the first 24 h, or the CHIR concentration was left at 3 µM but 10 µM of PI3K inhibitor LY294002 was added to the differentiation medium for the first 48 h.
3
Directed Cardiac Differentiation from hiPSCs
4
Maintenance of Mouse Embryonic Stem Cells
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mESCs were cultured at 37°C and 5% CO2 on gelatin-coated tissue culture plates/flasks in GMEM (Merck) supplemented with 10% embryonic stem cell qualified FBS (Gibco), GlutaMAX (Gibco), sodium pyruvate (Gibco), EmbryoMAX MEM NEAA (Merck), β-mercaptoethanol, or N2B27 (see below) supplemented with 3 μM CHIR99021 (Chir) (Stem Cell Technologies), 1 μM PD0305901 (Stem Cell Technologies) and 0.01 μg/ml LIF (Stem Cell Technologies). Cells were passaged every other day with Accutase (Merck) and maintained in culture for at least two passages post-thawing prior to experimental use. Cells were routinely tested for mycoplasma. If not stated otherwise, Sox1-GFP::Brachyury-mCherry cells53 (link) were used.
5
Cell Viability Assay with CHIR99021 and IWP-2
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WB-F344 cells were obtained from the Seoul National University Hospital (Seoul, Korea). The cells were cultured in Dulbecco's Modified Eagle's Medium F-12 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (WelGENE, Daegu, Korea) and 1% antibiotic-antimycotic (Gibco) at 37 in a 5% CO 2 incubator. Cells were cultured in 60-mm dish (0.6 × 10 6 cells) or 100-mm dish (1.5 × 10 6 cells) for 24 h and treated with 2-5 µM of CHIR99021 (CHIR, StemCell Technologies, Vancouver, Canada) for 24 h or 5-20 µM of IWP-2 (Sigma) for 48 h, and cell viability was detected using the CCK-8 kit (Dojindo, Tokyo, Japan). Briefly, the cells were seeded into a 96-well plate at a density of 5 × 10 3 cells/well for 24 h, treated with APAP in the presence or absence of CHIR or IWP-2, followed by the addition of CCK-8 reagent solution to each well and incubated for 2 h at 37 °C. Thereafter, the absorbance was measured at 450 nm using SpectraMax 190 (Molecular Devices, Sunnyvale, CA, USA).
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