Fluorescence-activated cell sorting of murine mammary cancer stem-like cell subpopulations with HIF activity

Danielle L. Brooks, Tiffany Seagroves

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Fluorescence-activated cell sorting (FACS) is a common method to identify and to isolate subpopulations within a complex mixture of cells based on their light scatter and fluorescent staining profiles. FACS is widely used to enrich for normal tissue and tumor cells that have stem cell potential. Whereas FACS protocols using conventional breast cancer cell lines are relatively routine, additional technical challenges are encountered when sorting for cell populations from freshly digested solid tumors, particularly for use in downstream cancer stem cell (CSC) assays. First, it is more difficult to isolate live, single cells from whole tumors, and second, single tumor cells prepared from enzymatically digested tumors are typically more sensitive to cell death following the physical stresses of digestion, pipetting, and sorting. Herein methods are described that have been optimized to harvest and to FACS profile viable tumor epithelial cells digested from late-stage mammary tumors originating in the mouse mammary tumor virus (MMTV)-polyomavirus middle T antigen (PyMT) transgenic mouse. Protocols were designed to enrich for single, viable, MMTV-PyMT tumor cell populations sorted by FACS and to facilitate the collection of sorted cell subpopulations suitable for head-to-head comparison of CSC activity by tumorsphere assays in vitro or limiting dilution transplantation in vivo.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages247-263
Number of pages17
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1742
ISSN (Print)1064-3745

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Neoplastic Stem Cells
Flow Cytometry
Breast Neoplasms
Neoplasms
Mouse mammary tumor virus
Polyomavirus Transforming Antigens
Colony-Forming Units Assay
Head and Neck Neoplasms
Complex Mixtures
Transgenic Mice
Population
Digestion
Cell Death
Stem Cells
Transplantation
Epithelial Cells
Staining and Labeling
Light
Cell Line

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Brooks, D. L., & Seagroves, T. (2018). Fluorescence-activated cell sorting of murine mammary cancer stem-like cell subpopulations with HIF activity. In Methods in Molecular Biology (pp. 247-263). (Methods in Molecular Biology; Vol. 1742). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7665-2_22

Fluorescence-activated cell sorting of murine mammary cancer stem-like cell subpopulations with HIF activity. / Brooks, Danielle L.; Seagroves, Tiffany.

Methods in Molecular Biology. Humana Press Inc., 2018. p. 247-263 (Methods in Molecular Biology; Vol. 1742).

Research output: Chapter in Book/Report/Conference proceedingChapter

Brooks, DL & Seagroves, T 2018, Fluorescence-activated cell sorting of murine mammary cancer stem-like cell subpopulations with HIF activity. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1742, Humana Press Inc., pp. 247-263. https://doi.org/10.1007/978-1-4939-7665-2_22
Brooks DL, Seagroves T. Fluorescence-activated cell sorting of murine mammary cancer stem-like cell subpopulations with HIF activity. In Methods in Molecular Biology. Humana Press Inc. 2018. p. 247-263. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7665-2_22
Brooks, Danielle L. ; Seagroves, Tiffany. / Fluorescence-activated cell sorting of murine mammary cancer stem-like cell subpopulations with HIF activity. Methods in Molecular Biology. Humana Press Inc., 2018. pp. 247-263 (Methods in Molecular Biology).
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