Normalized median fluorescence: an alternative flow cytometry analysis method for tracking human embryonic stem cell states during differentiation

Human embryonic stem cells (hESCs) are a promising cell source for tissue engineering and regenerative medicine, but before they can be used in therapies, we must be able to accurately identify the state and progeny of hESCs. One of the most commonly used methods for identification is flow cytometry. Many flow cytometry applications use antibodies to detect the amount of antigen present on/in a cell. This allows for the identification of unique cell populations or the tracking of expression changes within a population during differentiation. The results are typically presented as a percentage of positively expressing cells (%Pos) for a marker of choice, relative to a negative control. However, this reporting term is vulnerable to distortion from outliers and inaccuracy from loss of information about the population's fluorescence intensity. In this article, we describe an alternate strategy that uses the normalized median fluorescence intensity (nMFI), in which the MFI of the stained sample is normalized to the MFI of the negative control, as the reporting term to more accurately describe a population of cells in culture. We observed that nMFI provides a more accurate representation for the quality of a starting population and comparing data of different experimental runs. In addition, we demonstrated that the nMFI is a more sensitive measure of pluripotent and differentiation markers expression changes during hESC differentiation into three germ layer lineages.