Mouse ESCs and iPSCs were passaged in mouse ESC maintenance medium (DMEM, 15% fetal bovine serum [FBS; Gemini Bio], 0.1 mM non-essential amino acid [NEAA, Thermo Fisher Scientific], 0.1 mM β-mercaptoethanol [β-ME, Sigma-Aldrich], and 1,000 U/mL mouse LIF [ESGRO, EMD Millipore]) on gelatin-coated tissue culture plates as described previously (Liu et al., 2015 (link)). For EB differentiation, trypsinized ESCs were suspended in Costar ultra-low-attachment cell culture plates (Corning) at a density of 1 × 105 cells/ml in EB medium (DMEM, 15% Knockout Serum Replacement [Thermo Fisher Scientific], 0.1 mM NEAA, 0.1 mM β-ME) for 4 days. The EBs were seeded onto gelatin-coated plates at a density of 5 EB/cm2 and continued in culture in EB medium for 14 days. For nutlin-3a treatment, ESCs were differentiated in EB medium with 10 μM nutlin-3a (Cayman Chemicals). For inhibition of TGF-β and/or BMP signaling, ESCs were differentiated in EB medium containing 15 μM SB431542 (Sigma-Aldrich) and/or 250 ng/mL Noggin (PeproTech). EB medium was changed every other day.
Costar ultra low attachment cell culture plates
Manufactured by Corning
Costar ultra-low-attachment cell culture plates are designed for the cultivation of cells that have a tendency to grow in suspension or form spheroids. The plates feature a surface treatment that minimizes cell attachment, promoting the formation of three-dimensional cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Nutlin 3a, by Cayman Chemical (1 mentions)
Sb431542, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Noggin, by Thermo Fisher Scientific (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Non essential amino acid (neaa), by Thermo Fisher Scientific (1 mentions)
Esgro, by Merck Group (1 mentions)
Poly ornithine, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
2 protocols using costar ultra low attachment cell culture plates
1
Directed Differentiation of Mouse ESCs and iPSCs
2
Mouse ESC and iPSC Maintenance and Differentiation
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Mouse ESCs and iPSCs were maintained in mouse ESC maintenance medium (DMEM, 15% fetal bovine serum [FBS; Gemini Bio], 0.1 mM non-essential amino acid [Life Technologies], β-mercaptoethanol [Sigma-Aldrich], and 1,000 U/ml embryonic stem cell growth medium [ESGRO, Millipore]) on gelatin-coated plates as described previously (Kim et al., 2010 (link)). For EB differentiation, trypsinized wild-type or mutant iPSCs were suspended in Costar ultra-low-attachment cell culture plates (Corning) at a density of 1 × 105 cells/ml in differentiation medium (ESC maintenance medium without ESGRO). EB samples were collected on the indicated days for total RNA extraction. For neuronal differentiation, EBs (day 4) were plated onto tissue culture plates and cultured in N2 medium (DMEM/F12 and N2 supplement [Gemini Bio]) for up to 25 days. All fibroblasts were cultured in D10 medium (DMEM and 10% FBS). NSCs were cultured in Mouse Neural Stem Cell Expansion medium (EMD Millipore) on tissue culture plates coated with polyornithine (Sigma-Aldrich) and laminin (EMD Millipore).
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