G4 (C57BL/6Ncr x 129S6/SvEvTac) mouse hybrid23 (link) cells were maintained on STO feeder cells. For the experiment, they were subcloned and cultured on gelatinized plates (Corning) in N2B27 basal media (NDiff 227, StemCells) with addition of 100 U ml−1 of recombinant human leukemia inhibitory factor (Millipore), 1 μM of Mek1/2 inhibitor (PD0325901, Stemgent) and 3 μM of GSK3β inhibitor (CHIR99021, Stemgent). After three passages, when cells reached 75% confluence they were harvested by trypsinizing them with 0.05% trypsin/EDTA (Gibco).
Gelatinized plates
Manufactured by Corning
Gelatinized plates are a type of lab equipment designed to provide a stable and uniform surface for the cultivation of cells or tissues. They feature a thin layer of gelatin-based material applied to the surface, which enhances cell attachment and growth. These plates are commonly used in cell culture experiments and assays where a consistent and controlled environment is required.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Recombinant human leukemia inhibitory factor, by Merck Group (2 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Recombinant human lif, by Merck Group (1 mentions)
Cgp77675, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
3 protocols using gelatinized plates
1
Hybrid Mouse Embryonic Stem Cell Culture
2
G4 Embryonic Stem Cell Maintenance
3
Murine Embryonic Stem Cell Culture Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The G4 (C57BL/6Ncr x 129S6/SvEvTac) mouse hybrid (George et al., 2007 (link)) ESCs were obtained from Mount Sinai Hospital and were maintained on STO feeders in serum-containing media at 5% CO2 and 37°C. They were sub-cloned, and a line with normal karyotype was selected for further analysis. The cells were split onto gelatinized plates (10 cm, Corning) and expanded in serum-containing media or chemically defined media (standard 2i or alternative 2i) for at least three passages. Cells were harvested by trypsinization (0.05% trypsin/EDTA, GIBCO) for 10 min, at which point they reached 70%–80% confluence for single-cell capture.
The three media are as follows:
The three media are as follows:
Serum-containing media: Knockout DMEM (GIBCO), 1X penicillin-streptomycin-glutamine (GIBCO), 1X non-essential amino acids (GIBCO), 100 U/ml recombinant human leukemia inhibitory factor (Millipore), 15% fetal bovine serum (HyClone), 0.1mM β-mercaptoethanol (Sigma).
Standard 2i media: N2B27 basal media (NDiff 227, StemCells), 100 U/ml recombinant human LIF (Millipore), 1 μM PD0325901 (Stemgent), 3 μM CHIR99021 (Stemgent).
Alternative 2i media: N2B27 basal media (NDiff 227, StemCells), 100 U/ml recombinant human LIF (Millipore), 1 μM CGP77675 (Sigma), 3 μM CHIR99021 (Stemgent).
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