All mouse ESC lines were adapted for a minimum of 5 passages to growth in serum free N2B27 based medium supplemented with MEK inhibitor PD0325901 (0.8µM) and GSK3β inhibitor CHIR99021 (3.3µM) in tissue culture (TC) dishes pretreated with 7.5µg/ml polyL-ornithine (Sigma) and 5µg/ml laminine (BD) (Hayashi et al., 2011 ). To induce EpiLC differentiation, cells were washed with PBS, trypsinized and strained. 200,000 to 300,000 cells per 10cm2 were plated on TC dishes pretreated with 5µg/ml Fibronectin (Millipore) in N2B27 based medium supplemented with 1% KSR (Invitrogen) and 12µg/ml bFGF (Peprotech). Where indicated 20ng/ml Activin A (R&D Scientific) was added. For inhibition of all branches of the FGF signaling pathway, final concentration of 6.3µM FGF receptor tyrosine kinase inhibitor (EMD) was added to 2i+LIF medium.
Laminine
Manufactured by BD
Sourced in United States
Laminine is a laboratory equipment product. It is designed for use in various scientific and research applications. The core function of Laminine is to facilitate controlled and precise laboratory processes. Further details about its intended use or specific applications are not available.
Automatically generated - may contain errors
3 protocols using laminine
1
Establishment and Differentiation of Mouse ESCs
2
Serum-free mESC Culture Conditions
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mESCs were cultured on irradiated MEFs in N2B27 medium with 2i (PD0325901, 0.4 μM [Stemgent, San Diego, CA, USA] and CHIR99021, 3 μM [Stemgent]) and LIF (1000
u/ml) in tissue culture (TC) dishes pretreated with 7.5 µg/ml poly-L-ornithine (Sigma, St. Louis, MO, USA) and 5 µg/ml laminine (BD Biosciences, San Jose, CA,
USA) [26 (link)].
u/ml) in tissue culture (TC) dishes pretreated with 7.5 µg/ml poly-L-ornithine (Sigma, St. Louis, MO, USA) and 5 µg/ml laminine (BD Biosciences, San Jose, CA,
USA) [26 (link)].
3
Culturing male R1 mESCs in 2i + LIF
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R1 mESCs were cultured as previously described.66 (link) Briefly, male R1 cells were grown in monolayer on tissue culture plates pretreated with 7.5 ug/ml polyL-ornithine (Sigma) and 5 ug/ml laminine (BD) consecutively for 1 hour each in PBS. Cells were maintained in serum-free 2i + LIF media containing MEK inhibitor PD0325907 (0.8 uM), GSK3β inhibitor CHIR99021 (3.3 μM), and leukemia inhibitory factor (LIF).
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