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Understanding embryonic organogenesis and adult tissue regeneration relies on the ability to isolate and characterizing stem cells. However, these cells are difficult to study because they constitute minute populations in organ niches and express multiple cell-surface markers, few of which are identified. Furthermore, the properties of these cells change quickly in vitro and possibly even during isolation procedures. Perturbations introduced during sample isolation and processing, and the difficulty in determining gene-expression levels accurately in the low starting stem-cell numbers within the micro-anatomical niche further add further complications. Because they are better suited for large numbers of cells, fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) do not easily enable niche-specific characterization or even separate hind-leg muscle groups in the case of muscle (satellite) stem cells. Microscopy, although capable of imaging stem cells in their niches, is labor intensive and quantifying expression levels is difficult. Adding to the overall complexity is the fact that FACS, MACS, and fluorescence microscopy depend on irreversible antibody binding to stem-cell surface proteins, potentially altering cell properties, including gene expression and regenerative capacity. To address these challenges, we have developed a unique, label-free method for the objective, quantitative screening and characterization of single, functional organ stem cells. We demonstrate the power of our method by quantitatively screening satellite cells directly isolated from single muscle fibers.

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