Cells were cultured without feeders or serum, unless specifically noted, in N2B27 medium prepared as described2 (link) or preformulated (NDiff™ N2B27 base medium, Stem Cell Sciences Ltd, Cat. No. SCS-SF-NB-02) supplemented with Small molecule inhibitors PD0325901 (PD, 1 μM) and CHIRON99021 (CH, 3 μM) and LIF prepared in house (2i+LIF). Cells were routinely propagated on 0.1% gelatine-coated plastic and replated every 3 days at a split ratio of 1 in 10 following dissociation with Accutase (Gibco). Alternative Gsk3 inhibitors were provided by Pfizer: Compounds A (750 nM), B (50 nM), C (1 μM), D (250 nM), E (100 nM), F (150 nM) and G (50 nM) are described, see Supplementary Information , Figure 1 . Colony forming assays were performed by plating 600 ES cells per well on laminin (Sigma) coated plates. Plates were fixed and stained for alkaline phosphatase (Sigma) according to the manufacturer’s protocol. Plates were scanned using a CellCelector (Aviso) and scored manually.
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Serum-Free Mouse Embryonic Stem Cell Culture
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Accutase
Alkaline phosphatase
Assays
Cells
Es cells
Gelatine
Gsk3
Inhibitors
Laminin
Pd0325901
Serum
Stem cell
Corresponding Organization :
Other organizations : University of Cambridge, Wellcome/MRC Cambridge Stem Cell Institute, University College London, Max Planck Society, Max Planck Institute of Immunobiology and Epigenetics, Pfizer (United Kingdom)
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Variable analysis
independent variables
- Small molecule inhibitors PD0325901 (PD, 1 μM) and CHIRON99021 (CH, 3 μM)
- Alternative Gsk3 inhibitors: Compounds A (750 nM), B (50 nM), C (1 μM), D (250 nM), E (100 nM), F (150 nM) and G (50 nM)
- Colony forming assay: Plated 600 ES cells per well, fixed, stained for alkaline phosphatase, and scored manually
- Cells were cultured without feeders or serum, unless specifically noted
- Cells were routinely propagated on 0.1% gelatine-coated plastic and replated every 3 days at a split ratio of 1 in 10 following dissociation with Accutase (Gibco)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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