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Multiparameter flow cytometry for the characterization of human embryonic stem cells

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Abstract

Using multiparameter staining methods and flow cytometry to investigate the pluripotency of HUES7 human embryonic stem cell cultures, it was found that the multidimensional approach of marker co-expression allowed the different cell populations to be easily identified and demonstrated cross reactivity between the SSEA 4 and SSEA 1 antibodies, resulting in a substantial false positive SSEA 1 population. It is the accepted norm to apply control gates at a 95 % confidence level of the isotype control; however, this study found that adjusting the control gate to a 99 % confidence level significantly reduced the effect of this cross reactivity. Though conversely, this gating shift also decreased the positive marker expression of SSEA 4 and Tra-1-60, indicating that there is a need for strongly expressing markers coupled with increased optimization of fluorophore/antibody combinations before a gating strategy of 99 % can be implemented on a more routine basis.

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Acknowledgments

This work was funded as part of the Biotechnology and Biological Sciences Research Council (BBSRC; UK) Bioprocessing Research Industries Club (BRIC). We thank Nigel Smith, Department of Cytogenetics, Centre of Medical Genetics, Nottingham, UK for the karyotype analysis. We also greatly appreciate the assistance of Lorraine Young and Chris Denning, University of Nottingham, UK for providing the cells used and training in cell culture.

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Authors and Affiliations

  1. Centre for Biological Engineering, Department of Chemical Engineering, Loughborough University, Leicestershire, LE11 3TU, UK

    Kathryn Brosnan, Andrew Want, Karen Coopman & Christopher J. Hewitt

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  1. Kathryn Brosnan
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  2. Andrew Want
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  3. Karen Coopman
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  4. Christopher J. Hewitt
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Correspondence to Christopher J. Hewitt.

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Brosnan, K., Want, A., Coopman, K. et al. Multiparameter flow cytometry for the characterization of human embryonic stem cells. Biotechnol Lett 35, 55–65 (2013). https://doi.org/10.1007/s10529-012-1052-y

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  • DOI: https://doi.org/10.1007/s10529-012-1052-y

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