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Mouse Embryonic stem cells (ESCs) were isolated from mouse (Strain 129) embryos at Day E3.5. The cells were engineered to constitutively express EmGFP under the control of the human EF-1α promoter. Before cryopreservation, the cells were tested for viability (>70% post-thaw viability), their ability to remain undifferentiated when cultured on mouse feeder cells as assayed by marker expression (Oct4, SSEA-1, Nanog), their ability to differentiate into embryoid bodies and express markers indicative of three germ layers (Ectoderm: (+) for β3-tubulin and nestin, Mesoderm: (+) for smooth muscle actin, Endoderm: (+) for AFP) and expression of constitutive EmGFP as assayed by visual inspection of GFP fluorescence signal 10 days after thaw. Cells were cryopreserved at passage 30 (P30).
For optimal growth performance, we recommend culturing and expanding the cells in Knockout DMEM (SKU# 10829-018), Knockout serum replacement (SKU# 10828-028), Non-essential amino acids solution (SKU# 11140-050), L-glutamine (SKU# 25030081) and Recombinant human leukemia inhibitory factor (SKU# PHC9464).
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