12 well low attachment suspension culture plates

Single cells derived from mammary glands from transgenic or T47D cell lines were plated in 12-well low attachment suspension culture plates (Greiner Bio-One, Koln, Germany) at a density of 10,000 or 1000 viable cells/mL, respectively. Cells were grown in 1 mL serum-free media, supplemented with B27 (Gemini Bioproducts, West Sacramento, CA, USA), and 20 ng/mL EGF, as previously described [10 (link),30 (link)]. When indicated, mammosphere cultures were treated with 17-β-estradiol (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 10⁻⁸ M and ICI 182,780 (Santa Cruz Biotechnology, Dallas, TX, USA) at a concentration of 10⁻⁶ M. Both were prepared in absolute ethanol, which was used on its own as vehicle control. Mammospheres were counted after 7–10 days in culture with a Nikon eclipse TE2000-S inverted microscope. To calculate diameters and sphericity, mammosphere images were analyzed using Image J. Sphericity was calculated as longest diameter/shortest diameter; at least 15 mammospheres were measured per experiment and condition. Experiments were repeated at least 3 times.

Surviving cells after 3 days of BLM-gene treatments were trypsinized up to single cells and plated onto 12-well low attachment suspension culture plates (Greiner Bio- One) at a density of 2000 - 2500 viable cells/ml. Cells were grown in 1 ml serum-free media, supplemented with B27 (Gemini Bioproducts), and 20 ng/ml EGF. Melanospheres were counted after 6-8 days in culture. The melanosphere forming capacity was defined as the percentage of cells able of clonal proliferation as melanospheres with more than 8 cells.