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Limiting-Dilution Transplantation Assays in Mammary Stem Cell Studies

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Protocols for Adult Stem Cells

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 621))

Abstract

Mammary reconstitution assays can be used to measure the stem cell frequency within an epithelial population by transplanting increasingly diluted single-cell preparations of the population of interest. There are fundamental steps in the single-cell isolation protocol which are directly related to the number of single epithelial cells obtained. Once single-cell suspensions have been obtained, serial dilutions are prepared and transplanted into the cleared fat pads of the host mice. After 8–10 weeks, the transplanted fat pads are reevaluated for the presence of epithelial outgrowths. Based on the frequency of no outgrowth for each one of the transplanted dilutions, it is possible to estimate the frequency of mammary repopulating cells present in a given cell population. Here, we give details on how to carry out all these steps.

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Acknowledgments

This work was supported by a predoctoral fellowship to RFG from the Department of Defense Breast Cancer Research Program (DAMD 17-03-1-0594), grants from the same institution to COS (DAMD 17-00-1-0227 and DAMD 17-00-1-0306), a grant to BEW from the National Cancer Institute (CA 8424306), and a grant to MHBH funded by the National Institute of Environmental Health Sciences and the National Cancer Institute (U01 ES012801).

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Authors and Affiliations

  1. Langone School of Medicine, New York University, New York, NY, 10016, USA

    Irineu Illa-Bochaca & Mary Helen Barcellos-Hoff

  2. Development Biology Program, Sloan-Kettering, Institute, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA

    Rodrigo Fernandez-Gonzalez

  3. Department of Surgery, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, 84112, USA

    Dawne N. Shelton & Bryan E. Welm

  4. Morphology and Imaging Group and Cancer Imaging Laboratory, Center for Applied Medical Research, University of Navarre, Pamplona, 31008, Navarre, Spain

    Carlos Ortiz-de-Solorzano

Authors
  1. Irineu Illa-Bochaca
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  2. Rodrigo Fernandez-Gonzalez
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  3. Dawne N. Shelton
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  4. Bryan E. Welm
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  5. Carlos Ortiz-de-Solorzano
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  6. Mary Helen Barcellos-Hoff
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Editor information

Editors and Affiliations

  1. Dept. Bioengineering, University of California, Berkeley, Evans Hall 479, Berkeley, 94720-1762, USA

    Irina M. Conboy

  2. Helen Wills Neuroscience Inst., University of California, Berkeley, Stanley Hall 176, Berkeley, 94720-3220, USA

    David V. Schaffer

  3. Lawrence Berkeley National Lab., Dept. Cancer Biology, University of California, Berkeley, Cyclotron Rd. 1, Berkeley, 94720, USA

    Mary Helen Barcellos-Hoff

  4. Dept. Bioengineering, University of California, Berkeley, Stanley Hall, Berkeley, 94720-1762, USA

    Song Li

Appendix I

Appendix I

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Illa-Bochaca, I., Fernandez-Gonzalez, R., Shelton, D.N., Welm, B.E., Ortiz-de-Solorzano, C., Barcellos-Hoff, M.H. (2010). Limiting-Dilution Transplantation Assays in Mammary Stem Cell Studies. In: Conboy, I., Schaffer, D., Barcellos-Hoff, M., Li, S. (eds) Protocols for Adult Stem Cells. Methods in Molecular Biology™, vol 621. Humana Press. https://doi.org/10.1007/978-1-60761-063-2_2

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  • DOI: https://doi.org/10.1007/978-1-60761-063-2_2

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-062-5

  • Online ISBN: 978-1-60761-063-2

  • eBook Packages: Springer Protocols

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