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Abstract
Planarians possess remarkable stem cell populations that continuously support cellular turnover and are instrumental in the regeneration of tissues upon injury. Cellular turnover and tissue regeneration in planarians rely on the proper integration of local and systemic signals that regulate cell proliferation and cell death. Thus, understanding the signals controlling cellular proliferation and cell death in planarians could provide valuable insights for maintenance of adult body homeostasis and the biology of regeneration. Flow cytometry techniques have been utilized widely to identify, isolate, and characterize planarian stem cell populations. We developed alternative flow cytometry strategies that reduce the number of reagents and the time of sample preparation to analyze stem cells and cell death in planarians. The sensitivity of these methods is validated with functional studies using RNA interference and treatment with γ irradiation or stressful conditions that are known to trigger cell death. Altogether, we provide a community resource intended to minimize adverse effects during ex vivo studies of stem cells and cell death in planarians.
Highlights
Planarian flatworms are attractive model organisms for studies of stem cell biology, mechanisms of disease, maintenance of body homeostasis, and tissue regeneration (Aboobaker 2011; Reddien 2013; Rink 2013)
The initial FACS protocol adapted to planarian cells has been slightly modified over the years (Asami et al 2002; Reddien et al 2005; Hayashi et al 2006; Oviedo & Levin 2007; Eisenhoffer et al 2008; Fernandez-Taboada et al 2010; Pearson & Sanchez Alvarado 2010; Scimone et al 2010; Hayashi & Agata 2012; Moritz et al 2012; Peiris et al 2012; Romero et al 2012; Hubert et al 2013)
Our findings indicate that the gating strategy and FACS analysis involving DRAQ5 staining are effective to study diverse planarian cell populations
Summary
Planarian flatworms are attractive model organisms for studies of stem cell biology, mechanisms of disease, maintenance of body homeostasis, and tissue regeneration (Aboobaker 2011; Reddien 2013; Rink 2013). Planarians are able to regenerate missing parts in their body by activating adult somatic stem cells called neoblasts. Neoblasts are recognized as the only cells with proliferative capacity in planarians (Aboobaker 2011; Reddien 2013; Rink 2013). Planarians possess a tightly regulated system capable of controlling neoblast proliferation in response to the presence of tissue damage and localized cell death that ensures body homeostasis. Significant research attention is focused on understanding intrinsic neoblast properties and the signals regulating neoblast behavior
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