Cell proliferation assay

Breast cancer cells were plated in 96-well plates (black plate, clear bottom, Costar) at a low density (200–1000 cells/well in 100μl medium) to highlight solitary cell behavior. Indicated treatments were replenished every 3 days. HS5-CM was added to 30% final volume based on pilot titrations for maximal effect, except where stated otherwise; an equivalent volume of complete medium or 30% CM from other stromal lines as indicated was added to control wells. In pilot experiments, there was no difference between addition of complete medium or of MCF7-CM to experimental controls. Cell proliferation was measured at day 7 unless otherwise noted (for cells grown in complete growth medium) or day 14 (for cells in estrogen free medium) using a fluorescence-based cell proliferation kit (CyQUANT, Life Technologies) following the manufacturer’s instructions. These time points were optimal for a linear response of the cyquant assay when few cells are seeded. Fluorescence intensity (indicating cell numbers) was measured with a fluorescence 96-well plate reader (Beckman) at wavelength of 480 to 52 0nm. Each experimental replicate was done with 3–6 technical replicates.