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Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1

Abstract

Detecting and isolating specific types of cells is crucial to understanding a variety of biological processes, including development, aging, regeneration and pathogenesis; this understanding, in turn, allows the use of cells for therapeutic purposes, for which stem cells have emerged recently as invaluable materials. The current methods of isolation and characterization of stem cells depend on cell morphology in culture or on immunostaining of specific markers. These methods are, however, time consuming and involve the use of antibodies that may often make the cells unsuitable for further study. We recently developed a fluorescent small molecule named CDy1 (compound of designation yellow 1) that selectively stains live embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). This protocol describes detailed procedures for staining ESC and iPSC in live conditions and for fluorescence-activated cell sorting (FACS) of ESC using CDy1. Cell staining, image acquisition and FACS can be done within 6 h.

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Figure 1: Chemical structure and report of CDy1.
Figure 2
Figure 3: Isolating stem cells by CDy1 staining and FACS sorting.
Figure 4: Selective staining of mESC with CDy1.
Figure 5: hESC-BG01V characterization and staining with CDy1.
Figure 6: iPSC characterization and staining with CDy1.
Figure 7: Isolation of mESCs by FACS using CDy1.

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Acknowledgements

This study was supported by intramural funding from A*STAR (Agency for Science, Technology and Research, Singapore) Biomedical Research Council and a Young Investigator Award R-143-000-353-101 granted to Y.-T.C. from the National University of Singapore.

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Authors

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N.-Y.K. and S.-J.P. performed the experiments and processed the data. N.-Y.K. and S.-W.Y. wrote the manuscript. H.-H.H. synthesized CDy1 and analyzed its chemical properties. Y.-T.C. conceived, directed and supervised the project.

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Correspondence to Young-Tae Chang.

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The authors declare no competing financial interests.

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Kang, NY., Yun, SW., Ha, HH. et al. Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1. Nat Protoc 6, 1044–1052 (2011). https://doi.org/10.1038/nprot.2011.350

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