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A Detailed Mammosphere Assay Protocol for the Quantification of Breast Stem Cell Activity

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Since the discovery that neural tissue contains a population of stem cells that form neurospheres in vitro, sphere-forming assays have been adapted for use with a number of different tissue types for the quantification of stem cell activity and self-renewal. One tissue type widely used for stem cell investigations is mammary tissue, and the mammosphere assay has been used in both normal tissue and cancer. Although it is a relatively simple assay to learn, it can be difficult to master. There are methodological and analytical aspects to the assay which require careful consideration when interpreting the results. We describe here a detailed mammosphere assay protocol for the assessment of stem cell activity and self-renewal, and discuss how data generated by the assay can be analysed and interpreted.

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Cancer stem-like cells


Ductal carcinoma in situ


Oestrogen receptor


Epidermal growth factor


Invasive ductal carcinoma


Mammosphere forming efficiency


Pleural effusion


Progesterone receptor


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We would like to acknowledge the following funding sources: Breast Cancer Campaign (RBC and GF), Cancer Research UK (FLS), European Union FP6 (HH), European Union FP7 (KS and BMS), Biotechnology and Biological Sciences Research Council and Vertex Pharmaceuticals (Europe) Ltd. (MPA). We would like to thank Kate Williams for supplying the SUM-225 and micrographs.

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Correspondence to Robert B. Clarke.

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Frances L. Shaw and Hannah Harrison, are joint first authors

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Shaw, F.L., Harrison, H., Spence, K. et al. A Detailed Mammosphere Assay Protocol for the Quantification of Breast Stem Cell Activity. J Mammary Gland Biol Neoplasia 17, 111–117 (2012).

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