You are extracting protein from the cells or the conditioned medium?
A T25 flask has surface area of 25 cm2 and ~2 - 2.5 x 106 cells can be harvested at 100% confluence. MCF7 cells are recommended to be seeded at about 5 x 104 viable cells/cm2. The population doubling time is ~38 hours. In about 6 days you should be able to harvest about 2 x 106 cells.
Depending on the lysis/extraction method, you may obtain 1-10 ug total protein from 1 x 106 cells. How much protein you need for 2D depends on the 2D gel format (small or large) and detection method (silver stain is more sensitive than Coomassie blue ). For a large format gel (20 x 22 cm) a 100 ug load is recommended for silver and a 600 μg load for Coomassie blue staining.