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.
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Acknowledgments
This work was supported by the NIH (CA138488), the Department of Defense (IDEA award BC083846), and the UTHSC Gerwin Cancer Research fund to T.N.S. Luciana P. Schwab created the HIF-1 WT and KO PyMT tumor cell line models and developed initial flow sorting protocols. All experiments were conducted at the UTHSC Flow Cytometry and Flow Sorting (FCCS) core facility, which is supported by the UTHSC campus Office of Research. Expert technical assistance was provided by Drs. Tony Marion and Dan Rosson. The BD Biosciences Aria II sorter was purchased with the support of a NIH instrumentation award (S10 RR022465).
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Brooks, D.L., Seagroves, T.N. (2018). Fluorescence-Activated Cell Sorting of Murine Mammary Cancer Stem-Like Cell Subpopulations with HIF Activity. In: Huang, L. (eds) Hypoxia. Methods in Molecular Biology, vol 1742. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7665-2_22
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DOI: https://doi.org/10.1007/978-1-4939-7665-2_22
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