Tissue and malignancy stem cells are highly attractive target populations for regenerative medicine and novel potentially curative anticancer therapeutics. by circulation cytometry [9]. Allowing live cell recovery SP sorting is considered a valuable tool in stem cell research and has been successfully used to purify stem cells from diverse samples such as bone marrow tumor tissue and malignancy cell lines [10-15]. Traditionally SP analysis has Bcl-X been performed using the DNA-binding dye Hoechst 33342 [10]. Although this fluorophore works well and achieves excellent resolution it also requires an ultraviolet (UV) excitation source not commonly provided on standard circulation cytometers. Vybrant DyeCycle Violet (DCV) is usually another DNA-binding fluorophore suitable for SP detection that in contrast to Hoechst 33342 supports violet laser excitation thus enabling SP analysis of standard circulation cytometry instruments lacking a UV laser source [16]. Importantly the pump specificities of DCV and Hoechst 33342 are largely overlapping indicating that the same cell populations should be detected [16 17 However even though the DCV-based SP assay is usually increasingly used in stem cell research [14 18 the experimental parameters influencing the discrimination of DCV-SP cells have only been insufficiently elaborated. Similarly it is still a matter of argument which controls are most appropriate. Thus DCV-based SP detection GZ-793A is often performed under suboptimal conditions and/or without adequate controls each of which precludes the tapping of the method’s full potential. In this methods’ paper we systematically describe important experimental aspects of DCV-based SP detection. We propose that defined staining conditions as well as appropriate control selection is usually indispensable for the achievement of optimal results. In addition we depict common pitfalls and how to deal with them. Our paper should help scientists to optimize DCV-based SP detection for improved overall performance which is particularly relevant for the tiny stem cell populations present in tissues that inherently show a poor separation. Moreover we also view this paper as gateway for the standardization of this useful method for stem cell sorting. Finally we statement around the suitability of recently developed blue laser-excitable cell-permeant DNA dyes to discriminate ABC transporter-expressing cells. 2 Results 2.1 Theory and Workflow of SP Detection In SP detection cells of interest are loaded with a lipophilic DNA-binding fluorophore (e.g. DCV Hoechst 33342). Due to the membrane-permeant nature of such dyes they GZ-793A randomly enter all cells in the sample through passive diffusion and subsequently target GZ-793A nuclear and mitochondrial DNA for binding (Physique 1). The differential accumulation of such dyes between stem and nonstem cells constitutes the basis of SP detection: nonstem cells lacking ABC drug transporters retain high amounts of these dyes whereas stem cells efficiently prevent DNA-binding of the fluorophores by effluxing them right after entrance through mechanisms including functional drug transporter activity [9]. In addition DCV and Hoechst 33342 show a concentration-dependent switch in their emission spectra (“bathochromic shift”) that mechanistically results from electronic interactions between dye molecules bound to DNA. Accordingly when the sample is analyzed by circulation cytometry using bivariate dot plots for reddish and blue emission nonstem cells will have high fluorescence in both channels (due to the spectral shift particularly in the red channel) whereas the minority populace of stem cells will show dim fluorescence only appearing as the so-called SP in the lower left part of the plot (Physique 1). To finally confirm such low fluorescent cells asbona fideSP GZ-793A cells a functional inhibitor of ABC drug transporter activity (e.g. verapamil fumitremorgin C) needs to be included in the analysis and SP cells must disappear upon this pharmacological GZ-793A intervention [9]. Physique 1 Theory GZ-793A and workflow of SP detection. SP-defining dyes are lipophilic and enter cells passively to target nuclear and mitochondrial DNA. However binding to DNA occurs only in drug transporter-deficient (nonstem) cells whereas stem cells prevent this … It is important that this cells are adjusted to an defined cell count per mL and stained with an optimal amount of SP-defining dye (observe Physique 2). Because drug transporter-dependent dye.