A critical appraisal of factors affecting the accuracy of results obtained when using flow cytometry in stem cell investigations: Where do you put your gates?

Authors

  • Owen R. Hughes,

    1. Institute of Human Genetics and North East England Stem Cell Institute, University of Newcastle, International Centre for Life, Newcastle upon Tyne, United Kingdom
    Current affiliation:
    1. MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
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  • Rebecca Stewart,

    1. Institute of Human Genetics and North East England Stem Cell Institute, University of Newcastle, International Centre for Life, Newcastle upon Tyne, United Kingdom
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  • Ian Dimmick,

    Corresponding author
    1. Institute of Human Genetics and North East England Stem Cell Institute, University of Newcastle, International Centre for Life, Newcastle upon Tyne, United Kingdom
    • Institute of Human Genetics, University of Newcastle, Bioscience Centre, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ United Kingdom
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    • Ian Dimmick and Elizabeth A. Jones contributed equally to this work.

  • Elizabeth A. Jones

    1. Institute of Human Genetics and North East England Stem Cell Institute, University of Newcastle, International Centre for Life, Newcastle upon Tyne, United Kingdom
    Current affiliation:
    1. Genetic Medicine, Manchester Academic Health Science Centre, Central Manchester University Hospitals NHS Foundation Trust, Oxford Road, Manchester M13 9WL, United Kingdom
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    • Ian Dimmick and Elizabeth A. Jones contributed equally to this work.


Abstract

Flow cytometry is used extensively in stem cell investigations but there is wide variation in the methods used for data analysis between laboratories. Data analysis can be challenging in stem cell biology as there is often no clear distinction between positive and negative populations. We have undertaken a critical appraisal of factors that affect the accuracy of results in stem cell applications. We used mouse embryonic stem cells and determined the expression of three common antigens in stem cell investigations, namely CD15, CD184, and c-kit. We have compared different cell preparation methods and gating strategies and also evaluated the use of isotype controls and unstained cells as controls for the identification of positive populations. The use of a “doublet discriminator” using a side scatter area signal versus side scatter height signal dot plot to identify single cells for analysis increases the accuracy of results regardless of the method used to dissociate cells. Isotype controls can be helpful in mimicking cellular nonspecific binding of the experimental antibody reaction. Isotype controls behave differently on stem cells at different stages of differentiation. Analysis of a viable single cell population with careful selection of control cell populations increases the accuracy of results. © 2009 International Society for Advancement of Cytometry

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