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Limiting-Dilution Transplantation Assays in Mammary Stem Cell Studies

  • Irineu Illa-Bochaca
  • Rodrigo Fernandez-Gonzalez
  • Dawne N. Shelton
  • Bryan E. Welm
  • Carlos Ortiz-de-Solorzano
  • Mary Helen Barcellos-Hoff
Protocol
Part of the Methods in Molecular Biology™ book series (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.

Key words

Cell isolation Differential centrifugation Cleared fat-pad Transplantation Outgrowth Wholemount 

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Irineu Illa-Bochaca
    • 1
  • Rodrigo Fernandez-Gonzalez
    • 2
  • Dawne N. Shelton
    • 3
  • Bryan E. Welm
    • 3
  • Carlos Ortiz-de-Solorzano
    • 4
  • Mary Helen Barcellos-Hoff
    • 1
  1. 1.Langone School of MedicineNew York UniversityNew YorkUSA
  2. 2.Development Biology ProgramSloan-Kettering, Institute, Memorial Sloan-Kettering Cancer CenterNew YorkUSA
  3. 3.Department of SurgeryUniversity of Utah, Huntsman Cancer InstituteSalt Lake CityUSA
  4. 4.Morphology and Imaging Group and Cancer Imaging Laboratory, Center for Applied Medical ResearchUniversity of NavarrePamplonaSpain

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