Cell surface marker and cell cycle analysis, Hedgehog signaling, and flow cytometry

Detailed cytological analysis of cells undergoing differentiation often reveals clues to the regulation of multiple cell features. The Hedgehog (Hh) signaling cascade is a master regulator of cell fate during differentiation and is implicated in the development of some neoplasias. Hh signaling affects the expression of cell surface markers of differentiation. We have used the flow cytometer to evaluate the effect of blockage of the Hh signal on the expression of cell surface markers of erythroid differentiation in an in vitro system. In addition, the effect of Hh signaling on the distribution of cells in the phases of the cell cycle over the course of erythroid differentiation was assessed. Inhibition of the Hh signal retards progression of the erythroid developmental program. Included is a discussion of some of the basic parameters, limitations, and interpretations of flow cytometric analysis used for CD marker expression and cell cycle studies.