Monitoring the Cycling Activity of Cultured Human Keratinocytes Using a CFSE-Based Dye Tracking Approach

The development of methods and tools suitable for functional analysis of keratinocytes placed in an in vitro context is of great importance for characterizing properties associated with their normal state, for detecting abnormalities related to pathological states, or for studying the effects of extrinsic factors. In the present chapter, we describe the use of the intracellular fl uorescent dye carboxy fl uorescein succinimidyl ester (CFSE) to monitor cell division in mass cultures of normal human keratinocytes. We detail the preparation of CFSE-labeled keratinocyte samples and the identi fi cation by fl ow cytometry of cell subpopulations exhibiting different cycling rates in a mitogenic culture context. In addition, we show that the CFSE-based division-tracking approach enables the monitoring of keratinocyte responsiveness to growth modulators, which is here exempli fi ed by the cell-cycling inhibition mediated by the growth factor TGFβ 1. Finally, we show that keratinocyte subpopulations, separated according to their mitotic history using CFSE fl uorescence tracking, can be sorted by fl ow cytometry and used for further functional characterization, including determination of clone-forming ef fi ciency.