Multiparameter flow cytometry for the characterization of human embryonic stem cells

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.