@inbook{4445dfb5e07e4413afcb15fbe572c604,
title = "An RNA/DNA-Based Flow Cytometry Approach for Isolating Slow-Cycling Stem Cells",
abstract = "Flow cytometry methods for sorting specific populations of cells based on fluorescence or physical properties have been a widely used technique for decades. Flow cytometry has been particularly vital to the study of planarians, which remain refractory to transgenic transformation, as it has provided a work-around solution for studying stem cell biology and lineage relationships in the context of regeneration. Many flow cytometry applications have been published in planarians, beginning with broad Hoechst-based strategies for isolating cycling stem cells and progressing to more function-based approaches involving vital dyes and surface antibodies. In this protocol, we look to build on the classic DNA-labeling Hoechst staining strategy by adding pyronin Y staining to label RNA. While Hoechst labeling alone allows for the isolation of stem cells in the S/G2/M phases of the cell cycle, heterogeneity within the population of stem cells with 2 C DNA content is not resolved. By considering RNA levels, this protocol can further divide this population of stem cells into two groups: G1 stem cells with relatively high RNA content and a slow-cycling population with low RNA content, which we call RNAlow stem cells. In addition, we provide instruction for combining this RNA/DNA flow cytometry protocol with EdU labeling experiments and describe an optional step for incorporating immunostaining prior to cell sorting (in this case with the pluripotency marker TSPAN-1). This protocol adds a new staining strategy and examples of combinatorial flow cytometry approaches to the repertoire of flow cytometry techniques for studying planarian stem cells.",
keywords = "CellMask, EdU, Flow cytometry, Hoechst, Planarians, Pyronin Y, Stem cells",
author = "Nicole Lindsay-Mosher and Molinaro, {Alyssa M.} and Pearson, {Bret J.}",
note = "Publisher Copyright: {\textcopyright} 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.",
year = "2023",
doi = "10.1007/978-1-0716-3275-8_9",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "157--168",
booktitle = "Methods in Molecular Biology",
}