4. Gelatinized Plates: Using R1 ES cells, it is possible to carry out all ES cell manipulations on gelatinized plates from the initial passaging through to the introduction into mice. After replating cells from feeder containing plates onto gelatin plates, their characteristic morphology may be seen to change. This is usually only transient, and in time, they will revert to their usual appearance.
5. After mitomycin C treatment, the feeders are plated onto dishes for ES cell culture, the cells usually take an overnight incubation to attach they are then ready for use.
6. The medium should be changed from feeder cell medium (DMEM+ 10% FCS) to ES cell medium (DMEM+) before the addition of the ES cells.
7. An alternative to mitomycin C treatment is to treat the cells with 6000-10,000 rad of y-irradiation.
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