Two media formulations are used for ES cell culture. For ES cell isolation, medium contains 20% (v/v) serum (ES2o), whereas established ES cell lines may be maintained in medium with only 10% (v/v) serum (ES10) and supplemented with recombinant DIA/LIF (see Section 3.4.1. and Note 3). In both instances, serum comprises a 50:50 mixture of selected batches of fetal calf serum (FCS) and newborn calf serum (NCS).
1. Medium for murine embryonic stem cell isolation (ES20 medium)-64 mL Dulbecco's Modified Eagle's Medium (IX) 10 mL Fetal calf serum 10 mL Newborn calf serum 1 mL 200 mM L-glutamine 1 mL 100X nonessential amino acids stock 1 mL 100X (3-mercaptoethanol stock (final concentration: 0.1 mM) 13 mL sterile water Osmolality: ca. 290 mOsm/kgH20 (see Note 4).
2. Medium for murine embryonic stem cell maintenance (ES10 medium). 74 mL Dulbecco' s Modified Eagle' s Medium (IX) 5 mL Fetal calf serum 5 mL Newborn calf serum 1 mL 200 mM l-glutamine 1 mL 100X nonessential amino acids stock 1 mL 100X p-mercaptoethanol stock (final concentration: 0,1 mM) 13 mL sterile water Osmolarity: ca. 290 m0sm/kgH20 (see Note 4). To ES10 medium, BRL-conditioned medium or recombinant DIA/LIF may be added (see Section 3.4.1. and Note 3).
All media are filter-sterilized after preparation. Because of the large volumes concerned, it is often convenient to utilize resterilizable, 47mm filter holders (Millipore, Bedford, MA), housing a glass-fiber prefilter overlying a 0.22-|jm nitrocellulose filter. The medium is pushed through the filter with a 60-mL syringe. Media are stored at 4°C and warmed to 37°C immediately before use. Medium that is older than 2 wk should be supplemented with 1% (v/v) of 200 mM L-glutamine. Note that no antibiotics are used for ES cell isolation or culture (see Note 2).
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