Feeder-conditioned medium (feeder-CM)
Prepare mitomicin-treated mouse embryonic fibroblasts (MEF) (see Preparing Feeder Cell Layers from STO or Mouse Embryo Fibroblast (MEF) Cells: Treatment with Mitomycin C) and culture in TS medium for 72 hours. Collect the medium. Spin to remove the floating cells and debris, filter (0.45 μm), and store at -20°C in aliquots. Thaw each aliquot as needed and store it at 4°C; do not refreeze. Use the MEF to prepare two more batches of feeder-CM, then discard the cells. MEF are used up to 10 days after mitomycin treatment.
