Flow cytometric analysis is a valuable technique for identification and characterization of cells. However, flow cytometric analysis of autofluorescent cells requires careful consideration. In particular, the outcome of fluorescence compensation in the presence of significant autofluorescence poses a challenge to the interpretation of multicolour flow cytometric data. In this paper, we explain the mathematical basis of fluorescence compensation and the effects of autofluorescence, both in theory and applied to mesenchymal stem cells, which are notoriously autofluorescent. We illustrate how failure to understand the consequences of compensation can easily lead to critical errors in data interpretation, particularly when autofluorescence is involved. In the process, the common pitfalls of flow cytometric analysis of mesenchymal stem cells are presented, as are the simple measures necessary to avoid them. Specifically, the counterintuitive concept of "negative fluorescence values" is explained and exemplified, and the phenomenon of "population broadening" is addressed. Researchers must be acutely aware of the effects of compensation on the positioning of cells. We recommend always displaying data in biexponential or logicle transformations and advice to include fluorescence-minus-one controls to establish thresholds of positivity.
Keywords: Mesenchymal stromal cells, flow cytometry, cultured cells, cellular structures