Murine E14 ES cells were cultured on gelatin-coated plates in filtered cell medium consisting of Glasgow Minimum Essential Medium (GMEM; Invitrogen), 15% ES cell qualified fetal bovine serum (FBS; Invitrogen), 0.055 mM ß-mercaptoethanol (Sigma-Aldrich), 100 mM sodium pyruvate (Invitrogen), 0.1 mM MEM nonessential amino acid (NEAA, Invitrogen) and 1,000 units/ml of Leukemia Inhibitory Factor (LIF, Millipore). Platinum-E (Plat-E) cells were cultured in filtered Plat-E medium containing Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) and 10% FBS. SNL medium for maintenance of SNL feeder cells consists of GMEM and 10% FBS. AP staining was carried out using Alkaline Phosphatase Detection Kit (Millipore) according to the instructions provided in the kit. Stained colonies were viewed under an Axio Observer A1 inverted light microscope (Zeiss).
Pou5f1-GFP mouse embryonic fibroblasts (MEFs) infected with retroviruses for iPS cell formation were cultured in mES cell medium without LIF for 5 days post infection followed by maintenance in Knockout Serum Replacement (KSR) medium consisting of DMEM, 15% KSR (Invitrogen), 2 mM L- Glutamine (PAA), 1 mM sodium pyruvate, 1000 units/ml of LIF, 0.055 mM ß-mercaptoethanol and 0.1 mM MEM NEAA. All the cells were kept in a 37°C with 5% CO2 incubator.
Pou5f1-GFP mouse embryonic fibroblasts (MEFs) infected with retroviruses for iPS cell formation were cultured in mES cell medium without LIF for 5 days post infection followed by maintenance in Knockout Serum Replacement (KSR) medium consisting of DMEM, 15% KSR (Invitrogen), 2 mM L- Glutamine (PAA), 1 mM sodium pyruvate, 1000 units/ml of LIF, 0.055 mM ß-mercaptoethanol and 0.1 mM MEM NEAA. All the cells were kept in a 37°C with 5% CO2 incubator.