title: Multiparameter flow cytometry for the characterization of human embryonic stem cells.
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.
authors.affiliation: Centre for Biological Engineering, Department of Chemical Engineering, Loughborough University, Leicestershire, UK.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Brosnan
authors.forename: Andrew
authors.initials: A
authors.lastname: Want
authors.forename: Karen
authors.initials: K
authors.lastname: Coopman
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hewitt
mesh_terms: D000954:Antigens, Surface / Q000032:analysis / Q000737:chemistry; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000737:chemistry* / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016256:Lewis X Antigen / Q000032:analysis / Q000737:chemistry; D011509:Proteoglycans / Q000032:analysis / Q000737:chemistry; D055746:Stage-Specific Embryonic Antigens / Q000032:analysis / Q000737:chemistry
chemical_list: D000954:Antigens, Surface; D015415:Biomarkers; D016256:Lewis X Antigen; D011509:Proteoglycans; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; C066584:stage-specific embryonic antigen-4