Sigma-Aldrich Product Name ES-R1-ECFP CK8/ECFP, 07072005 biological source mouse embryo growth mode

Mouse embryonic stem cell, CFP expression

Pluripotent mouse embryonic stem cell line expressing cyan fluorescent protein (CFP). This cyan fluorescent variant was generated by the random integration of ECFP transgenes into the cell line ES-R1 (Sigma product number 07072001) using co-electroporation with a circluar selectable marker containing vector pPGK Puro. The vector is driven by a CMV immediate early  enhancer coupled to the chicken beta-actin promoter and first intron.

MEF medium consists of Advanced DMEM/F12, 10% FBS / FCS (F2442), 2 mM L-Glutamine (G7513) and 0.1 mM 2-Mercaptoethanol (M6250). KSR medium consists of DMEM, 20% Serum Replacer, 2 mM L-Glutamine (G7513), NEAA (M7145), 0.1 mM 2-Mercaptoethanol (M6250) and 1000 Units/ml LIF.

Embryonic stem (ES) cells require the use of mitotically inactivated feeder cells to support the growth of stem cells in the undifferentiated state. Mouse embryonic fibroblasts, STO (Sigma product number 86032003) or SNL 76/7 (Sigma product number 07032801) can be used. At ECACC plastic ware is pre-coated with gelatine prior to plating feeder cells.

Porcine gelatine (Sigma product number G1890) is dissolved in sterile water (0.5 g/500ml) at 56 °C. The 0.1% solution is sterilized by filtration (0.22 μm). Add 0.1% gelatine to plastic ware to cover bottom, and incubate for 20 minutes at room temperature (RT).  Remove gelatine, wash with PBS once and replace with appropriate culture medium. The flask/dish must not be allowed to dry out.

Feeder layers are prepared on the gelatinized flasks at least 24 hours in advance of being required. An ampoule is thawed in 37 °C water bath and the contents quickly transferred to a 15 ml centrifuge tube. MEF medium is added drop wise to 5 ml. Cells are centrifuged at 150 x g for 5 minutes at RT. Cells are resuspended in 5 ml of MEF medium. Cells are counted and added to flasks containing the correct medium at 1-3 x 10⁴ cells/cm².

An ampoule of ES cells is thawed in 37 °C water bath and the contents quickly transferred to a 15 ml centrifuge tube. KSR medium is added drop wise to 5 ml. Cells are centrifuged at 150 x g for 5 minutes. Cells are resuspended in 5 ml of KSR medium. The prepared feeder flask is washed once with PBS and KSR medium added. ES cells should be plated at 4-5 x 10⁴ cells/cm².  Cultures must be incubated in a humidified 5% CO₂/95% air incubator at 37 °C. A 100% media change must be performed every day and cells passaged every 2-3 days. Colonies must not be allowed to touch each other as overgrowth will result in differentiation.

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This cell line has special release conditions: Commercial organisations are required to complete the ′Cell Line Release Authorisation for Research Use in Commercial Organisations′ release conditions form.