ES-R1-EGFP B5/EGFP

mouse embryo

Adherent

Not specified

Adherent monolayer of spheroidal cells on feeder layer of mouse primary embryonic fibroblasts

Not specified

Not specified

cell culture | mammalian: suitable

dry ice

Mouse embryonic stem cell, GFP expression

Pluripotent mouse embryonic stem cell line expressing GFP. This green fluorescent variant was generated by the random integration of EGFP transgenes into ES-R1 (ECACC cat no. 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.

Not specified

MEF medium consists of Advanced DMEM/F12 (Invitrogen 12634010), 10% FBS (Perbio SH30070.03E), 2 mM Glutamine (Invitrogen 25030024) and 0.1 mM ß-mercaptoethanol (Sigma M6250).

KSR medium consists of KO-DMEM (Gibco 10829), 20% Knock-Out Serum Replacer (Gibco 10828), 2 mM Glutamine (Invitrogen 25030024), NEAA (Invitrogen 11140035), 0.1 mM ß-mercaptoethanol (Sigma M6250) and LIF 1000 Units/ml (ESGRO ESG1106).

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 (ECACC 86032003) or SNL 76/7 (ECACC 07032801) can be used. At ECACC plastic ware is pre-coated with gelatine prior to plating feeder cells.<p>Porcine gelatine (Sigma G1890) is dissolved in sterile water (0.5g/500ml) at 56^?C. The 0.1% solution is sterilized by filtration (0.22μm). Add 0.1% gelatine to plastic ware to cover bottom, and incubate for 20 minutes at room temperature.  Remove gelatine, wash with PBS once and replace with appropriate culture medium. <strong>The flask/dish must not be allowed to dry out.</strong>

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 15ml centrifuge tube. MEF medium is added drop wise to 5ml. Cells are centrifuged at 150 x g for 5 minutes at Room Temperature (RT). Cells are resuspended in 5ml 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 15ml centrifuge tube. KSR medium is added drop wise to 5ml. Cells are centrifuged at 150 x g for 5 minutes. Cells are resuspended in 5ml 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.