Primary mouse embryonic fibroblast (EMFI) cells (37) or the STO fibroblast cell line (38) is the most commonly used feeder layers. Details for the preparation of a stock of EMFI or STO cells are described elsewhere (39). A brief protocol for the preparation of EMFI feeder layers will be given here.
1. Quickly defrost a vial of EMFI or STO cells, and then transfer the cell suspension to a sterile 15-mL tube containing 10 mL prewarmed feeder cell media (DMEM supplemented with 10% FCS). Then spin at 1000g for 5 min, at room temperature.
2. Aspirate the supernatant, and then gently resuspend the cell pellet in 10 mL media.
3. Plate the cell suspension onto five 15-cm plates each containing 25 mL media, and place in an incubator.
4. When the cells form a confluent monolayer (usually takes 3 d) they are ready to be treated with mitomicin C.
5. Briefly, medium is aspirated from the confluent plates and replaced with 10 mL media containing 100 pL of 1 mg/mL mitomycin C, and then placed in an incubator for 2-2.5 h.
6. The medium is aspirated, and the plates washed twice each with 10 mL PBS, followed by the addition of 10 mL of trypsin/EDTA/dish.
7. Plates are placed in an incubator until the cells begin to detach.
8. Ten milliliters of media are added to each plate, and the cell suspension broken up by gentle pipeting.
9. The cell density is determined (hemocytometer) and adjusted to 2 x 105 cells/mL. Then the cells are plated directly onto dishes suitable for ES cell culture. We routinely plate approx 1 x 106 R1 cells per 10-cm dish (see Notes 5-7).
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