MEC were dissociated into single cells according to standard protocol and cultured in mammosphere media consisting of Epicult-B medium (Stem Cell Technology) containing 5% Matrigel, 10 ng/ml EGF (Sigma), 20 ng/ml bFGF (BD Biosciences), 4 μg/ml Heparin (Sigma), 5% heat inactivated FBS, 5 μM Rock inhibitor Y-27632 (Tocris) (24 (link)). Cells were seeded at 1,000-2,000 per well in 96-well ultralow attachment plates (Corning). The number of mammospheres (solid appearance, >50 um in diameter) was counted 7 days after seeding. Mammospheres were treated in vitro with 10 μg/ml TGFβ neutralizing antibody 1D11, IgG1 isotype control antibody, or 2 μM LY364947. Mammospheres pretreated for one week were dissociated and limiting dilutions were seeded into secondary passages without further treatment to measure self-renewal using L-Calc V1.1.1 (StemCell Technologies).
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Mammosphere Assay for Cancer Stem Cell Analysis
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Other organizations : New York University, University of California, San Francisco
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Variable analysis
independent variables
- TGFβ neutralizing antibody 1D11
- IgG1 isotype control antibody
- LY364947 (2 μM)
- Number of mammospheres (solid appearance, >50 um in diameter) counted 7 days after seeding
- Self-renewal of mammospheres measured by limiting dilutions in secondary passages
- Epicult-B medium (Stem Cell Technology) containing 5% Matrigel, 10 ng/ml EGF (Sigma), 20 ng/ml bFGF (BD Biosciences), 4 μg/ml Heparin (Sigma), 5% heat inactivated FBS, 5 μM Rock inhibitor Y-27632 (Tocris)
- Cell seeding density of 1,000-2,000 cells per well in 96-well ultralow attachment plates (Corning)
- IgG1 isotype control antibody
- Untreated mammospheres
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