Discussion

Stem cell biology constitutes one of the fastest growing areas in the life sciences. Accordingly, there is strong demand for improving the characterization tools and protocols available to stem cell researchers. We report here the development of a series of antibody-based tool sets and protocols that facilitate detection of important cellular markers of pluripotent stem cells and the differentiated cell types that can be derived from them (e.g., three germ layers, neural stem cells, cardiomyocytes). First, optimized immunocytochemistry reagent sets were identified by screening panels of validated primary antibodies against established stem cell markers and matching them up with appropriate dye-conjugated secondary antibodies and optimized fixative, permeabilization, blocking, and wash buffer systems. We demonstrate how these reagent sets can be applied to simplify traditional fixed-cell immunocytochemistry workflows and enable more information per sample via multiplex staining strategies that are compatible with a variety of imaging platforms. A second series of live-cell imaging reagents was generated to improve culture characterization and clone selection during cellular reprogramming workflows that are used to generate induced pluripotent stem cells. These tool sets are composed of dye-conjugated primary antibodies against select cell surface markers that are paired together with an imaging medium specifically designed to maximize fluorescence signal detection while maintaining cell health. We anticipate that this series of protocol and technology improvements will significantly augment the current characterization approaches available to stem cell researchers.