Development 6M4 cells

MDCK was cultured in a polystyrene culture flask (Nalge Nunc International, No136196) in the presence of 50 ^g/ml metalloproteinase (MEP) prepared from Streptomyces griseus, which received as a gift from Dr. S. Fujisaki (Institute of Enzyme Research, Osaka, Japan). The cells that were capable of growing in a suspension medium containing MEP were recovered and maintained as adherent cells (6M-4) in MEM-10 without MEP.

Colony selection was carried out by the dilution method on a 96 well polystylene-plate and by the selection of cells in soft agar. For the latter selection, cells were suspended in 15 ml of 0.3% agar, supplemented with complete growth medium. This cell suspension was allowed to solidify at room temperature on 10 ml of a 0.5% agar base layer containing growth medium in 10 cm dishes.

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